Interaction of Human Telomerase with Its Primer Substrate
Identifieur interne : 002229 ( Istex/Corpus ); précédent : 002228; suivant : 002230Interaction of Human Telomerase with Its Primer Substrate
Auteurs : Gerald Wallweber ; Sergei Gryaznov ; Krisztina Pongracz ; Ronald PruzanSource :
- Biochemistry [ 0006-2960 ] ; 2002.
Abstract
Telomerase is a ribonucleoprotein responsible for maintaining the ends of linear chromosomes in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line and progenitor cell populations. In the absence of telomerase activity, telomeres shorten with each cell division until a critical length is reached, which can result in the cessation of cell division. The enzyme is required for cell immortality, and its activity has been detected in the vast majority of human tumors. Because of this, telomerase is an attractive target for inhibition in anticancer therapy. To learn more about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have used highly purified human enzyme and employed a two-primer method for determining the dissociation rates of these primers. Primers having sequence permutations of (TTAGGG)3 were found to have considerably different affinities. They had t1/2 values that ranged from 14 min to greater than 1200 min at room temperature. A primer ending in the GGG register formed the most stable complex with the enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold decrease in the koff. We have found that interactions of telomerase with primer substrates are stabilized mainly by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primer and the template region of telomerase contributes minimally to its stabilization.
Url:
DOI: 10.1021/bi026914a
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ISTEX:D7E0CBE8517B444878A78467DFE6F3A10ED366A8Le document en format XML
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Phone: (650) 473-8631. E-mail: rpruzan@geron.com.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:D7E0CBE8517B444878A78467DFE6F3A10ED366A8</idno><date when="2003" year="2003">2003</date><idno type="doi">10.1021/bi026914a</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-P2TXNQ9N-G/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002229</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002229</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Interaction of Human Telomerase with Its Primer Substrate</title><author><name sortKey="Wallweber, Gerald" sort="Wallweber, Gerald" uniqKey="Wallweber G" first="Gerald" last="Wallweber">Gerald Wallweber</name><affiliation><mods:affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</mods:affiliation></affiliation></author><author><name sortKey="Gryaznov, Sergei" sort="Gryaznov, Sergei" uniqKey="Gryaznov S" first="Sergei" last="Gryaznov">Sergei Gryaznov</name><affiliation><mods:affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</mods:affiliation></affiliation></author><author><name sortKey="Pongracz, Krisztina" sort="Pongracz, Krisztina" uniqKey="Pongracz K" first="Krisztina" last="Pongracz">Krisztina Pongracz</name><affiliation><mods:affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</mods:affiliation></affiliation></author><author><name sortKey="Pruzan, Ronald" sort="Pruzan, Ronald" uniqKey="Pruzan R" first="Ronald" last="Pruzan">Ronald Pruzan</name><affiliation><mods:affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</mods:affiliation></affiliation><affiliation><mods:affiliation> Corresponding author. Phone: (650) 473-8631. E-mail: rpruzan@geron.com.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2002</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="589">589</biblScope><biblScope unit="page" to="600">600</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Telomerase is a ribonucleoprotein responsible for maintaining the ends of linear chromosomes in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line and progenitor cell populations. In the absence of telomerase activity, telomeres shorten with each cell division until a critical length is reached, which can result in the cessation of cell division. The enzyme is required for cell immortality, and its activity has been detected in the vast majority of human tumors. Because of this, telomerase is an attractive target for inhibition in anticancer therapy. To learn more about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have used highly purified human enzyme and employed a two-primer method for determining the dissociation rates of these primers. Primers having sequence permutations of (TTAGGG)3 were found to have considerably different affinities. They had t1/2 values that ranged from 14 min to greater than 1200 min at room temperature. A primer ending in the GGG register formed the most stable complex with the enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold decrease in the koff. We have found that interactions of telomerase with primer substrates are stabilized mainly by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primer and the template region of telomerase contributes minimally to its stabilization.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>primer</json:string><json:string>telomerase</json:string><json:string>koff</json:string><json:string>dissociation rate</json:string><json:string>biochemistry</json:string><json:string>koff value</json:string><json:string>htert</json:string><json:string>human telomerase</json:string><json:string>telomere</json:string><json:string>wallweber</json:string><json:string>dttp</json:string><json:string>datp</json:string><json:string>subunit</json:string><json:string>template region</json:string><json:string>room temperature</json:string><json:string>challenge primer</json:string><json:string>datum</json:string><json:string>euplotes</json:string><json:string>dgtp</json:string><json:string>nucleotide</json:string><json:string>guanosine</json:string><json:string>pruzan</json:string><json:string>telomerase activity</json:string><json:string>unpublished observation</json:string><json:string>nontelomeric primer</json:string><json:string>human enzyme</json:string><json:string>primer substrate</json:string><json:string>cell division</json:string><json:string>telomeric</json:string><json:string>analogue</json:string><json:string>assay</json:string><json:string>enzyme</json:string><json:string>dissociation</json:string><json:string>rate constant</json:string><json:string>bottom panel</json:string><json:string>guanosine triphosphate analogue</json:string><json:string>template antagonist</json:string><json:string>telomeric sequence</json:string><json:string>original primer</json:string><json:string>stable complex</json:string><json:string>anchor site</json:string><json:string>prism graphpad software</json:string><json:string>template</json:string><json:string>harley</json:string><json:string>shay</json:string><json:string>duplex</json:string><json:string>active site</json:string><json:string>ethylenediaminetetraacetic acid</json:string><json:string>primer substrate biochemistry</json:string><json:string>phosphorimager analysis</json:string><json:string>denaturing polyacrylamide</json:string><json:string>euplotes enzyme</json:string><json:string>protein subunit</json:string><json:string>cell free assay</json:string><json:string>stable interaction</json:string><json:string>dgtp analogue</json:string><json:string>bromophenol blue</json:string><json:string>telomeric primer</json:string><json:string>affinity</json:string></teeft></keywords><author><json:item><name>WALLWEBER Gerald</name><affiliations><json:string>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</json:string></affiliations></json:item><json:item><name>GRYAZNOV Sergei</name><affiliations><json:string>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</json:string></affiliations></json:item><json:item><name>PONGRACZ Krisztina</name><affiliations><json:string>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</json:string></affiliations></json:item><json:item><name>PRUZAN Ronald</name><affiliations><json:string>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</json:string><json:string>Corresponding author. 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To learn more about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have used highly purified human enzyme and employed a two-primer method for determining the dissociation rates of these primers. Primers having sequence permutations of (TTAGGG)3 were found to have considerably different affinities. They had t1/2 values that ranged from 14 min to greater than 1200 min at room temperature. A primer ending in the GGG register formed the most stable complex with the enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold decrease in the koff. We have found that interactions of telomerase with primer substrates are stabilized mainly by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primer and the template region of telomerase contributes minimally to its stabilization.</abstract><qualityIndicators><score>9.952</score><pdfWordCount>7660</pdfWordCount><pdfCharCount>46119</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>638</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>246</abstractWordCount><abstractCharCount>1623</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Interaction of Human Telomerase with Its Primer Substrate</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>42</volume><issue>2</issue><pages><first>589</first><last>600</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-P2TXNQ9N-G</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2003</publicationDate><copyrightDate>2003</copyrightDate><doi><json:string>10.1021/bi026914a</json:string></doi><id>D7E0CBE8517B444878A78467DFE6F3A10ED366A8</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-P2TXNQ9N-G/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-P2TXNQ9N-G/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-P2TXNQ9N-G/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-P2TXNQ9N-G/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Interaction of Human Telomerase with Its Primer Substrate</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2003 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2003</date><date type="Copyright" when="2003">2003</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Interaction of Human Telomerase with Its Primer Substrate</title><author xml:id="author-0000"><persName><surname>Wallweber</surname><forename type="first">Gerald</forename></persName><affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</affiliation></author><author xml:id="author-0001"><persName><surname>Gryaznov</surname><forename type="first">Sergei</forename></persName><affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</affiliation></author><author xml:id="author-0002"><persName><surname>Pongracz</surname><forename type="first">Krisztina</forename></persName><affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</affiliation></author><author xml:id="author-0003" role="corresp"><persName><surname>Pruzan</surname><forename type="first">Ronald</forename></persName><affiliation>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025</affiliation><affiliation role="corresp"> Corresponding author. Phone: (650) 473-8631. E-mail: rpruzan@ geron.com.</affiliation></author><idno type="istex">D7E0CBE8517B444878A78467DFE6F3A10ED366A8</idno><idno type="ark">ark:/67375/TPS-P2TXNQ9N-G</idno><idno type="DOI">10.1021/bi026914a</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2002</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="589">589</biblScope><biblScope unit="page" to="600">600</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>Telomerase is a ribonucleoprotein responsible for maintaining the ends of linear chromosomes
in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line
and progenitor cell populations. In the absence of telomerase activity, telomeres shorten with each cell
division until a critical length is reached, which can result in the cessation of cell division. The enzyme
is required for cell immortality, and its activity has been detected in the vast majority of human tumors.
Because of this, telomerase is an attractive target for inhibition in anticancer therapy. To learn more
about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding
properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have
used highly purified human enzyme and employed a two-primer method for determining the dissociation
rates of these primers. Primers having sequence permutations of (TTAGGG)<hi rend="subscript">3</hi> were found to have
considerably different affinities. They had <hi rend="italic">t</hi><hi rend="subscript">1/2</hi> values that ranged from 14 min to greater than 1200 min
at room temperature. A primer ending in the GGG register formed the most stable complex with the
enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold
decrease in the <hi rend="italic">k</hi><hi rend="subscript">off</hi>. We have found that interactions of telomerase with primer substrates are stabilized
mainly by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primer
and the template region of telomerase contributes minimally to its stabilization.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi026914a</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Interaction of Human Telomerase with Its Primer Substrate</article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Wallweber</surname><given-names>Gerald</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Gryaznov</surname><given-names>Sergei</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Pongracz</surname><given-names>Krisztina</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Pruzan</surname><given-names>Ronald</given-names></name><xref rid="bi026914aAF1">*</xref></contrib><aff>Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025
</aff></contrib-group><author-notes><corresp id="bi026914aAF1">
Corresponding author. Phone: (650) 473-8631. E-mail: rpruzan@
geron.com.
</corresp></author-notes><pub-date pub-type="epub"><day>17</day><month>12</month><year>2002</year></pub-date><pub-date pub-type="ppub"><day>21</day><month>01</month><year>2003</year></pub-date><volume>42</volume><issue>2</issue><fpage>589</fpage><lpage>600</lpage><history><date date-type="received"><day>27</day><month>09</month><year>2002</year></date><date date-type="rev-recd"><day>12</day><month>11</month><year>2002</year></date><date date-type="asap"><day>17</day><month>12</month><year>2002</year></date><date date-type="issue-pub"><day>21</day><month>01</month><year>2003</year></date></history><permissions><copyright-statement>Copyright © 2003 American Chemical Society</copyright-statement><copyright-year>2003</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Telomerase is a ribonucleoprotein responsible for maintaining the ends of linear chromosomes
in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line
and progenitor cell populations. In the absence of telomerase activity, telomeres shorten with each cell
division until a critical length is reached, which can result in the cessation of cell division. The enzyme
is required for cell immortality, and its activity has been detected in the vast majority of human tumors.
Because of this, telomerase is an attractive target for inhibition in anticancer therapy. To learn more
about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding
properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have
used highly purified human enzyme and employed a two-primer method for determining the dissociation
rates of these primers. Primers having sequence permutations of (TTAGGG)<sub>3</sub> were found to have
considerably different affinities. They had <italic toggle="yes">t</italic><sub>1/2</sub> values that ranged from 14 min to greater than 1200 min
at room temperature. A primer ending in the GGG register formed the most stable complex with the
enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold
decrease in the <italic toggle="yes">k</italic><sub>off</sub>. We have found that interactions of telomerase with primer substrates are stabilized
mainly by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primer
and the template region of telomerase contributes minimally to its stabilization.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi026914a</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="d7e144"><title></title><p>Human somatic cells have a finite proliferative capacity
that is limited by their telomere length (<italic toggle="yes"><xref rid="bi026914ab00001" ref-type="bibr"></xref></italic>). In the absence
of the enzyme telomerase, chromosome ends may be
shortened by dozens of nucleotides with each cell division
resulting from the inability of DNA polymerases to replicate
the ends of linear DNA (<italic toggle="yes"><xref rid="bi026914ab00002" ref-type="bibr"></xref></italic>). The consequences of telomere
shortening may be either a cessation of cell division or
chromosome instability and apoptosis when telomeres reach
a critically short length (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026914ab00003" ref-type="bibr"></xref>−<xref rid="bi026914ab00004" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026914ab00005" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026914ab00006" ref-type="bibr"></xref></named-content></italic>). Because of this, the activation
of telomerase can be linked to its ability to confer immortality
to cells, which has indeed been demonstrated for fibroblasts
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026914ab00007" ref-type="bibr"></xref>, <xref rid="bi026914ab00008" ref-type="bibr"></xref></named-content></italic>), retinal pigment epithelial cells (<italic toggle="yes"><xref rid="bi026914ab00007" ref-type="bibr"></xref></italic>), endothelial cells
(<italic toggle="yes"><xref rid="bi026914ab00009" ref-type="bibr"></xref></italic>), and other cell types (reviewed in ref <italic toggle="yes">10</italic>).
</p><p>Cells in the vast majority of human cancers express
telomerase whereas most normal somatic tissues lack expression. Cells that undergo malignant transformation necessitate
several independent genetic or epigenetic changes that
contribute to growth deregulation. Activation of telomerase
is required for cell immortality and is one of several events
that must occur for conversion of primary human cells into
tumor cells (<italic toggle="yes"><xref rid="bi026914ab00011" ref-type="bibr"></xref></italic>). These studies provided the rationale for
selecting telomerase as an attractive target for anticancer
therapy. The support for this approach was further buttressed
by the finding that overexpression of a dominant-negative
form of the enzyme could result in growth arrest or death of
tumor cell lines. The amount of time or number of cell
divisions necessary to reach crisis in a particular cell line
was related to the telomere length of that cell line (<italic toggle="yes"><xref rid="bi026914ab00012" ref-type="bibr"></xref></italic>).
Additional experiments with antisense oligonucleotides
directed against the template region of telomerase have
yielded similar results and support the key role of telomerase
in tumor cell maintenance (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026914ab00013" ref-type="bibr"></xref>−<xref rid="bi026914ab00014" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026914ab00015" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026914ab00016" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026914ab00017" ref-type="bibr"></xref></named-content></italic>).
</p><p>Telomerase is a ribonucleoprotein that utilizes a short
sequence within its RNA subunit as the template for reverse
transcription, synthesizing d(TG)-rich repeats onto the ends
of eukaryotic telomeres, which in vertebrates comprises the
hexanucleotide d(TTAGGG) (<italic toggle="yes"><xref rid="bi026914ab00018" ref-type="bibr"></xref></italic>). In vitro, telomerase
catalyzes the addition of d(TTAGGG) repeats onto a single-stranded DNA primer. For the human enzyme, two telomerase components are necessary and sufficient for this activity
in vitro: the previously mentioned RNA (hTR)<xref rid="bi026914ab00001" ref-type="bibr"></xref> and a protein
that constitutes the catalytic subunit (hTERT). We have been
interested in a rational approach to finding telomerase
inhibitors as potential therapeutics and recently described
several oligonucleotide phosphoramidates and thio-phosphoramidates addressed to the RNA component of the enzyme
(<italic toggle="yes">17</italic>, <italic toggle="yes">19</italic>, <italic toggle="yes">20</italic>). These oligonucleotides have picomolar to low
nanomolar potency in inhibiting telomerase activity in cell
free assays, suggesting very efficient template antagonism.
Further knowledge of human telomerase, especially its
substrate recognition, should allow us to better select reagents
as potential competitive inhibitors of the enzyme.
</p><p>In cells, the single-stranded DNA 3‘-overhang at the end
of the telomere, d(TTAGGG)n, is the natural substrate for
the enzyme. Short single-stranded oligonucleotides that have
minimal complementary to the template region of telomerase
can serve as primers in cell free assays (<italic toggle="yes"><xref rid="bi026914ab00021" ref-type="bibr"></xref></italic>). In this work,
we have begun to investigate the interactions of human
telomerase with its DNA primer substrate and have measured
the relative affinities of several primers for the enzyme. We
found that while enzyme−primer complexes can be stabilized
by base-pairing with the hTR template as well as by
interactions between hTERT and the 5‘-end of the primer,
an additional strong interaction occurs between hTERT and
the 3‘-end nucleotides of the primer.
</p></sec><sec id="d7e220"><title>Materials and Methods</title><p><italic toggle="yes">Purification of Human Telomerase.</italic> Telomerase was
prepared from 293 suspension cells that overexpressed the
hTERT gene, using a myeloproliferative sarcoma virus
promoter. Whole cell extracts were prepared from frozen cell
pellets. The cell pellets were resuspended in one packed cell
volume of H buffer (10 mM <italic toggle="yes">N</italic>-2-hydroxyethylpiperazine-<italic toggle="yes">N</italic>‘-2-ethanesulfonic acid-KOH (pH 7.9), 10 mM KCl, 1 mM
MgCl<sub>2</sub>, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
floride, and 0.5 μg/mL Leupeptin) and then lysed with a
dounce homogenizer. The concentration of salt in the lysate
was adjusted to 0.3 M NaCl, which was stirred for 15 min
and then centrifuged at 100 000<italic toggle="yes">g</italic>. Solid ammonium sulfate
was added to the supernatant to achieve 42% saturation, and
insoluble proteins were pelleted. The pellets were resuspended in A buffer (20 mM <italic toggle="yes">N</italic>-2-hydroxyethylpiperazine-<italic toggle="yes">N</italic>‘-2-ethanesulfonic acid-KOH (pH 7.9), 1 mM MgCl<sub>2</sub>, 1 mM
dithiothreitol, 1 mM ethyleneglycoltetraacetic acid, 10%
glycerol, and 0.5 μg/mL Leupeptin) containing 0.1 M NaCl
(1/5 of their original volume) and dialyzed against this buffer.
Following dialysis, the extract was spun at 25 000<italic toggle="yes">g</italic> to remove
insoluble material. Telomerase was purified by antisense
affinity chromatography similar to previously described
methods, using a 2‘-O-Me RNA complementary to the
template region of hTR (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026914ab00022" ref-type="bibr"></xref>, <xref rid="bi026914ab00023" ref-type="bibr"></xref></named-content></italic>). Affinity purified extracts
were further purified by size-exclusion chromatography
(G5000PW, Tosoh Biosep LLC). The amount of telomerase
in the extracts was determined by quantitative northern blot
analysis of hTR and by quantitative measurement of the
telomerase activity. The activity measurement was conducted
by pulse-labeling a saturating amount of primer (100 nM
18AGG, Table <xref rid="bi026914at00001"></xref>) with 20 μCi [α-<sup>32</sup>P] dGTP (3000 Ci/mmol)
for 30 min at 22 °C, which permitted the addition of a single
nucleotide. Under the conditions used, there was no substrate
turnover during the pulse-labeling, and thus, we were able
to measure the moles of active enzyme in the preparation.
The number of active enzyme molecules found agreed with
the number of hTR molecules determined by northern
analysis in the preparation, which was between 0.1 and 0.2
pmol of telomerase per milliliter of extract, and we thus
inferred an equal stiochiometry between hTR and hTERT.
<table-wrap id="bi026914at00001" position="float" orientation="portrait"><label>1</label><caption><p>Primers Used for Binding Studies</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"><graphic xlink:href="bi026914au00001a.tif" position="float" orientation="portrait"></graphic></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Contains 3‘-NHP(O)(S-)O-5‘ thio-phosphoramidate (NPS) linkages
and a 3‘-terminal amino group.</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Determination of Dissociation Rates of Primers. </italic>Extracts
containing telomerase (G5000PW pool, 0.5 fmol/20 μL
reaction) were incubated with stated primers (100 nM) at
22 °C for 30 min in telomerase reaction buffer that contained
50 mM (<italic toggle="yes">N</italic>-2-hydroxyethylpiperazine-<italic toggle="yes">N</italic>‘-3 propanesulfonic
acid)-NaOH (pH 8.5), 1 mM MgCl<sub>2</sub>, 1 mM dithiothreitol,
5% glycerol, and 0.5 mM ethyleneglycoltetraacetic acid.
Primer−telomerase complexes were then challenged by the
addition of 20 μM 24GGG or 18GGG (Table <xref rid="bi026914at00001"></xref>). At times
indicated in the figures, aliquots (18 μL) were removed and
added to a nucleotide mix (2 μL) containing 2 mM dTTP
and 10 μCi [α-<sup>32</sup>P] dATP (3000 Ci/mmol) and labeled for 5
min. The reactions were terminated with sodium dodecyl
sulfate (0.1%), NH<sub>4</sub> acetate (750 mM), and pellet paint (2
μL, Novagen) in a final volume of 200 μL. The terminated
reactions were extracted with an equal volume of phenol-chloroform (1:1) and precipitated with 2.5 volumes of
ethanol. Following centrifugation, the pellets were washed
with 80% ethanol and resuspended in 90% formamide (10
μL) containing bromophenol blue and xylene cylanole. The
samples were applied to a 20% polyacrylamide gel containing
7 M urea, 115 mM tris(hydroxymethyl) aminomethane, 15
mM boric acid, and 0.6 mM ethylenediaminetetraacetic acid
and electrophoresed at 21 W until the bromophenol blue
migrated off the gel. Following electrophoresis, the gel was
fixed in 5% trichloroacetic acid (w/v), rinsed in water, dried,
and analyzed with a PhosphorImager (Molecular Dynamics).
</p><p>For primers ending in GGG, GGT, or GTT, the rate
constants were determined by following the disappearance
of the labeled primer. Each primer is expressed as percent
bound relative to the zero time point. Primers ending in TTA,
TAG, or AGG are not extended with the dTTP and [α-<sup>32</sup>P]
dATP nucleotide mix, so rate constants were determined by
following the appearance of the labeled 24GGG challenge
primer. For this set of primers, percent bound is determined
by first taking the difference from the maximum at each time
point. In either case, the rate constants were determined by
fitting the data to the equation <italic toggle="yes">y</italic> = <italic toggle="yes">A</italic> exp(−<italic toggle="yes">kt</italic>) (where <italic toggle="yes">A</italic>
represents the amount of primer in complex at time zero, <italic toggle="yes">k</italic>
is the rate constant, and <italic toggle="yes">t</italic> is the time in minutes), using the
Prism software package (GraphPad Software Inc.).
</p><p><italic toggle="yes">Dissociation Rates of Primers Having a 3</italic>‘<italic toggle="yes">-Terminal
Guanosine Analogue. </italic>All modified primers are derivatives
of the 18GGG (Table <xref rid="bi026914at00001"></xref>) sequence that terminate with a
guanosine analogue. Primers ending in a 2‘-deoxy-(dG), 3‘-deoxy, ribo-, or 2‘,3‘-dideoxyguanosine (ddG) were synthesized using standard solid-phase chemistry with the appropriate column, followed by purification through a 15%
polyacrylamide gel containing 7 M urea. The primer was
identified by UV shadowing, excised, eluted into 0.5 M NH<sub>4</sub>
acetate (pH 8.0) and 1 mM ethylenediaminetetraacetic acid,
and purified through a C-18 Sep-Pak cartridge (Waters Corp).
Primer−telomerase complex formation, labeling, and processing of reactions were identical as described above.
</p><p>Primers ending in 3‘-amino- or 3‘-azido- were generated
by incubating 25 pM telomerase and 100 nM primer 18AGG
(Table <xref rid="bi026914at00001"></xref>), with an additional 250 μM of the guanosine
triphosphate analogue, for 30 min at 22 °C. Challenging,
labeling, and processing of reactions were identical as
described above. To verify that the guanosine triphosphate
analogues were incorporated, two additional reactions containing 25 pM telomerase were incubated with 20 μM of
the 24GGG primer for 30 min at 22 °C. For each, an 18-μL
aliquot was removed and labeled with a 2-μL nucleotide mix
containing 2 mM dTTP and 10 μCi [α-<sup>32</sup>P] dATP (3000 Ci/mmol; NEN) alone or with the additional 250 μM guanosine
triphosphate analogue for 5 min. Quantification of the +3
and +4 products showed that greater than 98% of the labeled
primer incorporated the guanosine triphosphate analogue.
</p><p><italic toggle="yes">Competition between Primer 18GGG and Thio-Phosphoramidate GRN163. </italic>Telomerase containing extract (G5000PW
pool, 0.5 fmol/20 μL reaction) was incubated with 100 nM
primer 18GGG in telomerase reaction buffer (see above) for
30 min at 22 °C. Complexes were then challenged by the
addition of 0.5−50 nM thio-phosphoramidate GRN163
(Table <xref rid="bi026914at00001"></xref>). After an additional 30-min incubation, 18-μL
aliquots were removed, labeled, and processed as described
above. The products were separated on an 18% (1:19 bis-acrylamide) polyacrylamide gel containing 7 M urea, 115
mM tris(hydroxymethyl) aminomethane, 15 mM boric acid,
and 0.6 mM ethylenediaminetetraacetic acid. The gel (140
× 170 × 0.8 mm) was electorphoresed at 21 W until the
bromophenol blue migrated off the gel. The gel was fixed
in 5% trichloroacetic acid (w/v), rinsed in water, dried, and
analyzed with a PhosphorImager (Molecular Dynamics).
</p></sec><sec id="d7e343"><title>Results</title><p>We have purified human telomerase from 293 cells that
overexpress the catalytic (hTERT) subunit of telomerase.
These cells were found to possess about 5-fold more activity
than the parental 293 cells (unpublished observation, R.
Pruzan). Starting with whole cell extracts, we have purified
telomerase using an affinity selection step (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026914ab00022" ref-type="bibr"></xref>, <xref rid="bi026914ab00023" ref-type="bibr"></xref></named-content></italic>) followed
by size-exclusion chromatography. The resultant material was
enriched approximately 10 000-fold for telomerase. The only
enzymatic activity in the extracts capable of labeling an
exogenous primer was deemed as telomerase by virtue of
its RNA dependence, its sensitivity to a highly specific
antitemplate telomerase inhibitor oligophosphoramidate
GRN163 (17, Figure <xref rid="bi026914af00005"></xref>), as well as the correctly predicted
products generated by incubating the purified extract along
with primers having different 3‘-ends with the appropriate
[α-<sup>32</sup>P] dNTP (data not shown and Figure <xref rid="bi026914af00002"></xref>).
</p><p>The affinity of an enzyme for its substrate can be assessed
from its dissociation constant (<italic toggle="yes">K</italic><sub>D</sub>). The <italic toggle="yes">K</italic><sub>D</sub> for a given
substrate can be determined by varying its concentration and
measuring the amount bound to enzyme as a function of its
concentration. Alternatively, the dissociation rate (<italic toggle="yes">k</italic><sub>off</sub>) of
enzyme bound substrates can be used to determine relative
affinities of the substrates for the enzyme. Measuring the
<italic toggle="yes">k</italic><sub>off</sub> is often a much more experimentally tractable approach
and was chosen when determining primer affinities for
telomerase. We developed a primer-binding assay that
depended on telomerase activity, which provided us with a
highly specific assay, since only primers bound to the active
site of the enzyme were tallied. Initially, a saturating amount
of the primer of interest was incubated with enzyme. After
reaching equilibrium, the enzyme−primer complex was
challenged with a large excess of a competitive primer having
a different length than the original one. Aliquots were
removed at various time intervals and pulse-labeled with
dTTP and [α-<sup>32</sup>P] dATP, which resulted in either the original
primer or the challenge primer producing a discrete end-labeled product. These products were then resolved by
denaturing polyacrylamide gel electrophoresis and analyzed
by PhosphorImager scanning. The amount of labeled products were normalized to the amount of starting material at
time zero and expressed graphically, which in turn was used
to derive dissociation rates for the various primers.
</p><p>When the association rate of the primer with enzyme (and
the catalytic rate) is much greater than the dissociation rate,
either the disappearance of the extended original primer with
time, or conversely, the appearance of the extended challenge
primer can serve to monitor the dissociation rate of the primer
(Figure <xref rid="bi026914af00001"></xref>C). This property allowed us to examine the binding
of primers that could not be extended, for example, those
lacking a 3‘-OH terminal group. Employing the same
challenge primer allowed us to measure a variety of different
primers using a consistent set of conditions. The technique
is illustrated in Figure <xref rid="bi026914af00001"></xref>, where the intensity of the band
representing the product of the initial enzyme−primer
complex decreases as a function of time. A concomitant
increase in the band representing the product of challenge
primer is also seen (Figure <xref rid="bi026914af00001"></xref>A,B). The amount of product
resulting from the initial enzyme−primer complex (21-mer
in Figure <xref rid="bi026914af00001"></xref>A and 27-mer in Figure <xref rid="bi026914af00001"></xref>B) is assigned the value
of 100% (time zero). The amount of complex remaining at
subsequent times is expressed as a fraction of that initial
amount. The 100% value for the challenge primers product
is obtained by pulse-labeling a preequilibrated mixture of
the initial primer and challenge primer (200-fold excess of
the latter). When telomerase is prebound with the 18GGG
primer (see Table <xref rid="bi026914at00001"></xref> for sequence of primers) and challenged
with the 24GGG primer, pulse-labeled products 3-nt longer
than the original primers result from the addition of TTA.
Thus, the 18GGG and 24GGG primers are converted into
21- and 27-nt products, respectively. Identical results were
obtained when the band intensities of the 21- or the 27-nt
products are quantitated individually and used to derive the
dissociation rate of 18GGG (Figure <xref rid="bi026914af00001"></xref>C). This is noteworthy
since some of the primers used in our study cannot be labeled
using dTTP and [α-<sup>32</sup>P] dATP, thus only the appearance of
the challenge primer is visible. Furthermore, when the
reciprocal experiment is done, first incubating telomerase
with 24GGG followed by a challenge with an excess of
18GGG (Figure <xref rid="bi026914af00001"></xref>B,D), an equivalent rate of dissociation for
24GGG was observed, indicating that 18GGG is already
longer than the minimal length requirement necessary to
stabilize its interaction with the enzyme and that additional
nucleotides at the 5‘-end of the primer do not provide any
added stability.
<fig id="bi026914af00001" position="float" orientation="portrait"><label>1</label><caption><p>Assay for measuring dissociation rate of primers from telomerase. Telomerase containing extract (20 μL) was incubated with primer (100 nM) for 30 min at 22 °C in a total reaction volume of 200 μL as described in Materials and Methods. The mix was equilibrated at 37 °C after which the challenge primer (20 μM) was added. Aliquots (18 μL) were removed at times indicated and added to a nucleotide mix (2 μL) containing 10 μCi [α-<sup>32</sup>P] dATP and 2 mM dTTP and pulse-labeled for 5 min. The samples were extracted and analyzed on
a 20% denaturing polyacrylamide gel as described in Materials and Methods. (A) Enzyme incubated with 18GGG and challenged with
24GGG. (B) Enzyme incubated with 24GGG and challenged with 18GGG. (C) Bands depicted in panel A were quantitated by PhosphorImager
analysis and expressed as a percentage of complex bound based on the sample in which the challenge primer was premixed with the initial
primer. (D) Data from experiments depicted in panels A and B were analyzed as in panel C, and dissociation rates were determined from
the graphs using the Prism GraphPad software.</p></caption><graphic xlink:href="bi026914af00001.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Dissociation Rates of Permutated Telomeric Primers.
</italic>Human telomerase is able to extend a variety of primers with
different efficiencies in cell free assays (<italic toggle="yes"><xref rid="bi026914ab00021" ref-type="bibr"></xref></italic>), although the
elements involved in their recognition have not been well-defined. We have begun to investigate the determinants of
primer recognition and specificity using the two-primer
method described above. Using 24GGG as the competitor,
we measured the <italic toggle="yes">k</italic><sub>off</sub> of six different 18-nt telomeric primers,
representing the six possible permutations of a repetitive
TTAGGG sequence. The sequences of these primers are
listed in Table <xref rid="bi026914at00001"></xref>, and the dissociation rates for these primers
from telomerase can be seen in Figure <xref rid="bi026914af00002"></xref>. These data are
presented graphically along with a summary of the derived
<italic toggle="yes">k</italic><sub>off</sub> values for these primers (Figure <xref rid="bi026914af00002"></xref>G). When telomerase
is incubated with 18GGG, 18GGT, and 18GTT, in the
presence of dTTP and [α-<sup>32</sup>P] dATP, the enzyme will
generate products of 21-, 20-, and 19-nt, respectively (Figure
<xref rid="bi026914af00002"></xref>A−C). Primers 18TTA, 18TAG, and 18AGG cannot be
labeled with dTTP and [α-<sup>32</sup>P] dATP (Figure <xref rid="bi026914af00002"></xref>D−F).
Therefore, the appearance of the 27-nt band originating from
the labeling of the competitor primer 24GGG was used for
determining the <italic toggle="yes">k</italic><sub>off</sub> values for this group of primers. The
comparison between the six permutated 18-mers was carried
out at room temperature (∼22 °C) to capture those primers
that had rapid dissociation rates. The <italic toggle="yes">k</italic><sub>off</sub> values ranged from
0.047 min<sup>-1</sup> for the least stable 18TTA to <0.00058 min<sup>-1</sup>
for the most stable 18GGG (Figure <xref rid="bi026914af00002"></xref>A−G). The dissociation
rate of the latter primer was not measurable at room
temperature. It was followed for 20 h, after which a half-life for this primer−telomerase complex was still not reached
(data not shown); hence, we ascribe a rate of <0.00058
min<sup>-1</sup>, which represents a <italic toggle="yes">t</italic><sub>1/2</sub> of >20 h. Three of the primers
(18GTT, 18AGG, and 18TAG) had similar <italic toggle="yes">k</italic><sub>off</sub> values of
about 0.02 min<sup>-1</sup> (Figure <xref rid="bi026914af00002"></xref>, panels C, E−G), and 18GGT
had an intermediate <italic toggle="yes">k</italic><sub>off</sub> (0.002 min<sup>-1</sup>) that was about 10-fold slower than the previous three (Figure <xref rid="bi026914af00002"></xref>C−G). Thus,
the exact 3‘-register of the TTAGGG hexanucleotide repetitive sequence is extremely important for its recognition by
the enzyme. Telomeric primers can theoretically form
between five and 11 base-pairs with the RNA template of
telomerase (Figure <xref rid="bi026914af00003"></xref>A). A primer terminating in TAG can
be depicted as fully aligned with the template in a pretranslocation register or partially aligned, poised to be extended
(ref <italic toggle="yes">18</italic>, Figure <xref rid="bi026914af00003"></xref>A). Dissociation rates therefore do not
correlate with the number of potential base-pairs that a primer
can form with the template. It is noteworthy that 18TTA,
which can potentially form the most complete duplex with
the RNA template, has the most rapid <italic toggle="yes">k</italic><sub>off</sub> (<italic toggle="yes">t</italic><sub>1/2</sub> ∼15 min,
Figure <xref rid="bi026914af00002"></xref>G). Thus, significant interactions apart from base-pairing with the template must be involved in stabilizing the
interaction between primer and enzyme. Given the identical
length of all six primers, one trivial explanation for the rapid
dissociation rate of 18TTA was the loss of a potential
stabilizing contact between the 5‘-end of the primer and
hTERT. As depicted in Figure <xref rid="bi026914af00003"></xref>A, 18TTA would align with
the 5‘-end of the template region, which could potentially
distance its 5‘-end from a putative binding site on hTERT.
To rule out this possibility, two additional primers, 19TTA
and 20TTA (Table <xref rid="bi026914at00001"></xref>), were synthesized. These primers
should maintain their 3‘-nucleotide alignment with the
template, while having the identical 5‘-end as 18GTT and
18GGT, which have respective 2- and 20-fold lower dissociation rates as compared to 18TTA (Figure <xref rid="bi026914af00002"></xref>G). Only a
minor decrease in the <italic toggle="yes">k</italic><sub>off</sub> was observed (less than 2-fold)
when the 5‘-end of 18TTA was extended (Figure <xref rid="bi026914af00003"></xref>B−D).
Taken together, these data indicate that a telomerase−primer
complex may be stabilized by an intricate set of interactions;
some that may reside at or near the 3‘-end of the primer.
The extremely stable complex observed with 18GGG and
the rapid change in stability, with respect to a single
nucleotide change of the register of the primer, would be
compatible with the 3‘-terminus having a different topology
in relation to the hTERT protein domain.
<fig id="bi026914af00002" position="float" orientation="portrait"><label>2</label><caption><p>Dissociation rates of the six permutated sequences of primer (TTAGGG)<sub>3</sub>. Telomerase containing extract (20 μL) was incubated
with primer (100 nM) for 30 min at 22 °C in a total reaction volume of 200 μL as described in Materials and Methods. Following the
addition of the 24GGG challenge primer (20 μM), aliquots (18 μL) were removed and added to a nucleotide mix (2 μL) containing 10 μCi
[α-<sup>32</sup>P] dATP and 2 mM dTTP and pulse-labeled for 5 min. The samples were extracted and analyzed on a 20% denaturing polyacrylamide
gel as described in Materials and Methods. The following primers were initially incubated with telomerase in panels A−F, 18GGG, 18GGT,
18GTT, 18TTA, 18TAG, and 18AGG. (G) Bands depicted in panels A−F were quantitated by PhosphorImager analysis and expressed as
a percentage of complex bound based on the sample in which 24GGG was added together with 18-nt primer. <italic toggle="yes">k</italic><sub>off</sub> values for primers are
depicted in panel G as determined from the graphs using the Prism GraphPad software.</p></caption><graphic xlink:href="bi026914af00002.tif" position="float" orientation="portrait"></graphic></fig><fig id="bi026914af00003" position="float" orientation="portrait"><label>3</label><caption><p>Primer's affinity for telomerase depends on its register with respect to the template. Dissociation rates for primers were determined as described in Materials and Methods and in legend to Figure <xref rid="bi026914af00002"></xref>. (A) Schematic of the template region of hTR is depicted together with primers having the six possible sequence permutations of (TTAGGG)<sub>3</sub> and their possible alignment with the template. (B) Dissociation of
19TTA. (C) Dissociation of 20TTA. (D) Bands depicted in panels B and C were quantitated by PhosphorImager analysis and expressed as
a percentage of complex bound based on the sample in which 24GGG was added together with 19- or 20-nt primer. Also shown is the
dissociation rate of 18TTA taken from Figure <xref rid="bi026914af00002"></xref>G. <italic toggle="yes">k</italic><sub>off</sub> values for primers depicted in panel D are determined from the graphs using the
Prism GraphPad software.</p></caption><graphic xlink:href="bi026914af00003.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Sites of Telomerase−Primer Interactions Are Separable.
</italic>Two sites of primer interaction with telomerase have been
previously proposed: an anchor site interacting with the 5‘-end of the primer and a template site interacting with the
3‘-end of the primer (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026914ab00021" ref-type="bibr"></xref>, <xref rid="bi026914ab00024" ref-type="bibr"></xref></named-content></italic>). Data supporting this model
was obtained by experiments photocross-linking a primer
with the <italic toggle="yes">Euplotes </italic>enzyme (<italic toggle="yes"><xref rid="bi026914ab00025" ref-type="bibr"></xref></italic>).
</p><p>To discern the different contributions to the primer
complex stability in the human enzyme, we compared the
<italic toggle="yes">k</italic><sub>off</sub> values of four primers, 18GGG, CH18GGG, NT18GGG,
and NT18GTT (Table <xref rid="bi026914at00001"></xref>). As mentioned above, 18GGG
forms an extremely stable complex with telomerase at room
temperature. NT18GTT is an 18-nt nontelomeric primer
ending with GTT-3‘ (Table <xref rid="bi026914at00001"></xref>) that can be extended by
telomerase. This primer is used in the common TRAP assay
(<italic toggle="yes"><xref rid="bi026914ab00026" ref-type="bibr"></xref></italic>). It binds only weakly to the enzyme, having a <italic toggle="yes">k</italic><sub>off</sub> of
about 0.4 min<sup>-1</sup> (Figure <xref rid="bi026914af00004"></xref>A). In fact, we found NT18GTT
to bind telomerase with an affinity similar to a random
oligomer, when monitoring the end-labeled primer's ability
to bind the enzyme using a nitrocellulose filter-binding assay
(data not shown). Primer 18GTT (Table <xref rid="bi026914at00001"></xref>), by contrast, has
a <italic toggle="yes">k</italic><sub>off</sub> of about 0.02 min<sup>-1</sup> (Figure <xref rid="bi026914af00002"></xref>G). This affinity difference
may be attributed to its ability to form additional base-pairs
with the template region or to a stronger interaction with
the anchor site of hTERT.
<fig id="bi026914af00004" position="float" orientation="portrait"><label>4</label><caption><p>Nontelomeric primer ending in GGG has a high affinity for telomerase. Dissociation rates for primers were determined as described in Materials and Methods and in the legend to Figure <xref rid="bi026914af00002"></xref>. Comparison of dissociation rates of NT18GTT (A), NT18GGG (B), or CH18GGG (C) (Table <xref rid="bi026914at00001"></xref>). (D) Graphical representation of dissociation rates from panels A−C together with that of 18GGG from Figure <xref rid="bi026914af00002"></xref>G.<italic toggle="yes">k</italic><sub>off</sub> values for primers depicted in panel D as determined from the graphs using the Prism GraphPad software.
</p></caption><graphic xlink:href="bi026914af00004.tif" position="float" orientation="portrait"></graphic></fig></p><p>To separate the stability contributions of the primer's
register from those of either the template or the anchor site
interaction, we compared the affinity of three primers ending
in the GGG-3‘ register. We designed a primer (NT18GGG,
Table <xref rid="bi026914at00001"></xref>) identical to NT18GTT that ended with TGGG-3‘
instead of AGTT-3‘. This primer is not complementary to
the template region with the exception of the last three G's.
The A was converted to T to eliminate one extra base-pair.
NT18GGG was found to dissociate from telomerase with a
<italic toggle="yes">k</italic><sub>off</sub> of 0.0048 min<sup>-1</sup> (Figure <xref rid="bi026914af00004"></xref>B,D). Thus, similar to primers
with a telomeric sequence, the register ending with GGG-3‘
confers a dramatic increase to the enzyme−primer stability.
CH18GGG was designed as a chimeric primer, 11 5‘-nucleotides are isosequential to NT18GTT, and seven 3‘-nucleotides matched those of 18GGG. It is fully complementary to the seven 3‘-nucleotides of the hTR template
region. Replacing four nucleotides of NT18GGG with ones
complementary to the template reduces the <italic toggle="yes">k</italic><sub>off</sub> of the primer
between 2−3-fold (Figure <xref rid="bi026914af00004"></xref>B−D), which indicates that most
of the potential additional base-pairs are likely not formed.
Comparing the <italic toggle="yes">k</italic><sub>off</sub> values for 18GGG and CH18GGG reveals
the former is at least 3-fold more stable than the latter
(<0.00058 and 0.0018 min<sup>-1</sup>, respectively; Figure <xref rid="bi026914af00004"></xref>D),
indicating a preference for telomeric sequences at the 5‘-end of the primer. While TTAGGG-based telomeric sequences contribute to the stability of the telomerase−primer
complex (10-fold or greater reduction in <italic toggle="yes">k</italic><sub>off</sub> ), adding stability
through contacts at both the 5‘-end and through an interaction
with the template, the alignment of the 3‘-end of the primer
in the active site represents a previously unidentified and
potentially more significant interaction in providing stability
to the complex. Substituting acyclovir or 3‘-azido-dG for the
3‘-ultimate nucleotide in 18GGG destabilizes the complex
that the primer forms with the enzyme at room temperature,
such that <italic toggle="yes">k</italic><sub>off</sub> values 0.0068 and 0.0022 min<sup>-1</sup> are observed
respectively (data not shown). While the former modification
affects both base-pairing and stacking interactions, the
reduced stability of the latter substitution suggests a disruption of an interaction in the sugar domain of the nucleoside
since 3‘-azido-dG can base-pair similar to a 3‘-OH-dG.
</p><p><italic toggle="yes">18GGG Competes with hTR Template Antagonist GRN163.
</italic>The stability of a primer−telomerase complex depends on
the aggregate of elaborate interactions between the enzyme
and the primer. We have compared the binding of 18GGG,
whose 3‘-terminal nucleotide register was found to have the
most stable interaction with telomerase, to that of an
extremely potent and specific telomerase template antagonist,
GRN163. The latter is a 13-nt thio-phosphoramidate oligonucleotide complementary to nucleotides 42−54 of hTR that
is a competitive inhibitor of telomerase, having an IC<sub>50</sub> value
of 0.4 nM (<italic toggle="yes"><xref rid="bi026914ab00017" ref-type="bibr"></xref></italic>). When GRN163 was prebound to purified
telomerase, it prevented binding and labeling of 18GGG in
a dose-dependent manner (Figure <xref rid="bi026914af00005"></xref>, lanes 5−8). If however,
the order of addition was reversed, 18GGG was prebound
to the enzyme, and GRN163 was added, then the complex
was refractory to binding and inhibition by GRN163 (Figure
<xref rid="bi026914af00005"></xref>, lanes 1−4). This demonstrates the very stable nature of
the 18GGG primer complex and supports our finding that
these two oligonucleotides compete at least partially for the
same binding site (data not shown). GRN163 is a hTR
template antagonist, and it interacts primarily with hTR,
whereas 18GGG is stabilized significantly by interactions
with the protein component of telomerase. This was demonstrated by binding either <sup>32</sup>P-labeled 18GGG or <sup>32</sup>P-labeled
GRN163 to telomerase and comparing the migration of the
resultant complexes formed on a native gel (unpublished
observation). While both the primer and the template
antagonist labeled a complex that migrated similar to an
endogenously labeled telomerase complex, treatment of the
GRN163-labeled complex with proteinase K resulted in a
shift of that complex to a position on the gel similar to that
of labeled hTR, whereas the same treatment of an 18GGG-labeled complex resulted in the disappearance of any primer
complex (unpublished observation).
<fig id="bi026914af00005" position="float" orientation="portrait"><label>5</label><caption><p>18GGG primer prevents binding of template antagonist GRN163. Purified telomerase (0.5 fmol) was preincubated with the telomeric primer 18GGG (100 nM) for 30 min at room temperature and then challenged with GRN163 at concentrations that varied from 0.5 to 50 nM (lanes 1−4). In lanes 5−8, the enzyme was first incubated with GRN163 for 10 min, after which primer 18GGG was added. The enzyme complex was pulse-labeled for 5 min with dTTP (200 μM) and [α-<sup>32</sup>P] dATP (10 μCi, 3000 Ci/mmol).</p></caption><graphic xlink:href="bi026914af00005.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Effect of 3</italic>‘<italic toggle="yes">-Terminus Modifications on Primer Binding.
</italic>To explore potential interactions between the 3‘-end of the
primers and telomerase, we have examined the <italic toggle="yes">k</italic><sub>off</sub> values
of several primers ending in GGG-3‘ in which the sugar
residue of the terminal nucleoside was modified. Because
of the high stability of the complex formed between
telomerase and 18GGG, we were unable to define dissociation rates at room temperature and have therefore conducted
the experiments comparing the primers with modified sugars
at 37 °C. At that temperature, 18GGG has a <italic toggle="yes">k</italic><sub>off</sub> of 0.013
min<sup>-1</sup> (Figure <xref rid="bi026914af00006"></xref>A,G). We tested the effect of having either
an H or an OH in the 2‘- or 3‘-positions. A primer with a
2‘-OH and a 3‘-H was found to have a modest increase in
its <italic toggle="yes">k</italic><sub>off</sub> (0.031 min<sup>-1</sup>; Figure <xref rid="bi026914af00006"></xref>B,G). This difference could
either indicate a stabilizing influence of a 3‘-OH group or a
destabilizing one of a 2‘-OH group. We then tested the
binding of a primer having hydroxyl groups at both the 2‘
and 3‘-positions and found it to have a nearly identical <italic toggle="yes">k</italic><sub>off</sub>
as the previous primer, 0.034 min<sup>-1</sup>, which indicated the 2‘-OH has a modest destabilizing effect on the binding of the
primer (Figure <xref rid="bi026914af00006"></xref>C,G). This conclusion was confirmed by
determining the <italic toggle="yes">k</italic><sub>off</sub> of a terminal dideoxy primer, which had
stability similar to 18GGG (0.013 min<sup>-1</sup>; Figure <xref rid="bi026914af00006"></xref>D,G). We
also surveyed two additional 3‘-modifications: a 3‘-azido
and a 3‘-amino. Both of these terminal modifications were
created by incubating telomerase with 18AGG followed by
the addition of either 3‘-NH<sub>2</sub>-dGTP or 3‘-N<sub>3</sub>-dGTP (250 μM).
While both of these nucleotides are chain terminators and
thus do not allow labeling of the original primer, we can
infer the analogues were readily incorporated at the end of
the original primer as the challenge primer (24GGG) was
extended to a 28-nt pulse-labeled product as compared to a
27-mer that was seen when the nucleotide triphosphate was
absent (Figure <xref rid="bi026914af00006"></xref>E,F). At the same time we observed,
however, that the presence of a dGTP analogue in the
reaction mixture reduced the affinity of 18GGG by approximately 2-fold (data not shown) and have adjusted the
<italic toggle="yes">k</italic><sub>off</sub> values for these two primers to reflect this fact (Figure
<xref rid="bi026914af00006"></xref>G, bottom panel). At 37 °C the destabilizing effect of the
azido substitution is similar to the 2‘-OH substitution, 2−3-fold (Figure <xref rid="bi026914af00006"></xref>G, bottom panel). In contrast, the 3‘-amino
modified primer was moderately stabilized (approximately
4-fold) relative to its OH counterpart (Figure <xref rid="bi026914af00006"></xref>G, bottom
panel). The more basic nitrogen atom in the 3‘-position
appears able to form an additional interaction with the protein
subunit. We surmised that the additional stability of the 3‘-amino primer was a consequence of the basicity of nitrogen.
We tested this supposition by measuring the effect that
acetylating the 3‘-amino group had on the primer's affinity.
Acetylation increased the <italic toggle="yes">k</italic><sub>off</sub> to about 0.014 min<sup>-1</sup>, which
was similar to that of 18GGG (data not shown). This supports
the notion that the nucleophilic electron pair of the nitrogen
is responsible for the more stable interaction of the 3‘-amino
primer. Thus, while the exact interaction between the 3‘-end of 18GGG and the enzyme is still undefined, it is clear
that 2‘ or 3‘ groups that affect charge, nucleophilicity, and
steric requirements can all influence the primer's affinity,
and thus, the stability of a primer in this register results from
an aggregate of interactions.
<fig id="bi026914af00006" position="float" orientation="portrait"><label>6</label><caption><p>Dissociation rates of primers terminating in GGG register having modifications in their terminal sugar moiety. Dissociation rates for the indicated primers were determined as described in Materials and Methods and in the legend to Figure <xref rid="bi026914af00002"></xref> except that experiments were conducted at 37 °C. The 3‘-NH<sub>2</sub> and 3‘-N<sub>3</sub> primers were generated by incubating telomerase and 18AGG (100 nM) with the respective
nucleotide triphosphates (250 mM) for 30 min at room temperature. Lanes A and B represent reactions where 24GGG (20 μM) was added
along with the 18AGG primer without or with the addition of nucleotide triphosphate, respectively. The <italic toggle="yes">k</italic><sub>off</sub> of 18GGG was increased
approximately 2-fold in the presence of guanosine nucleoside triphosphates, and hence the <italic toggle="yes">k</italic><sub>off</sub> reported for the 3‘-NH<sub>2</sub> and 3‘-N<sub>3</sub> primers
was adjusted in the table of panel G to reflect this fact and is indicated in the bottom panel of panel G (*).
</p></caption><graphic xlink:href="bi026914af00006.tif" position="float" orientation="portrait"></graphic></fig></p></sec><sec id="d7e740"><title>Discussion</title><p>Telomerase is a ribonucleoprotein that catalyzes the
synthesis of TTAGGG repeats onto the ends of
chromosomes. The template for these repeats is an intrinsic part of
the RNA subunit of the enzyme. In vitro, the enzyme can
use short single-stranded DNAs as the substrate for the
repeated nucleotide addition. We have determined the <italic toggle="yes">K</italic><sub>m</sub>
for the 18-nt primer, (TTAGGG)<sub>3</sub> (at 37 °C), to be about 2
nM, which indicates a highly specific interaction with this
substrate (unpublished data, R. Pruzan). We were interested
in examining the interactions between telomerase and the
substrate primer that contribute to this specificity. A binding
assay was established that depended on pulse-labeling the
primers, which had the advantage of recording only primers
bound to the enzyme's active site. We measured the
dissociation rates of 18-nt primers that had the six permutated
sequences of TTAGGG. The most interesting finding was
the nearly 100-fold range in <italic toggle="yes">k</italic><sub>off</sub> values that were observed
between the different primer registers of TTAGGG repeats.
A primer ending in GGG-3‘ formed a complex with
telomerase that was stable at room temperature (<italic toggle="yes">t</italic><sub>1/2</sub> greater
than 20 h) as compared to one ending in TTA-3‘ that
dissociated from the enzyme with a <italic toggle="yes">t</italic><sub>1/2</sub> of about 15 min.
The differences in affinity could neither be attributed to
differences at the 5‘-end of the primer nor solely to changes
in base-pairing with the template. While the added potential
GC base-pair for 18GGG versus 18AGG could potentially
explain the added stability (−2.1 kcal mol<sup>-1</sup>; ref <italic toggle="yes">28</italic>) for the
former primer, that trend is not followed when subsequent
nucleotides are added at the 3‘-end of the primer (Figure
<xref rid="bi026914af00002"></xref>G).
</p><p>Primers 18GGT and 20TTA have identical 5‘-ends, and
20TTA has the potential to form two additional base-pairs,
yet 18GGT has a 15-fold higher affinity for the enzyme.
Thus, it appears that the entire template is not involved with
base-pairing to the primer and that interactions between the
primer and the protein subunit hTERT are likely the key
determining factors involved in stabilizing the enzyme−primer complex. A further indication that there is limited
base-pairing between the template of hTR and a primer
substrate came from data comparing the primers NT18GGG
with CH18GGG. While the latter primer has the potential
to form four additional base-pairs with the template of hTR,
only a modest decrease in the <italic toggle="yes">k</italic><sub>off</sub> (2−3-fold) was observed
for CH18GGG, suggesting that base-pairing has only a minor
role in primer stabilization. Energetically, these four additional base-pairs would be expected to result in greater than
a 2000-fold increase in stability (∼-4.6 kcal mol<sup>-1</sup>; ref <italic toggle="yes">28</italic>);
thus, they are most likely not occurring. Previous studies
with <italic toggle="yes">Euplotes</italic> telomerase have indicated that the entire
template is not used for base-pairing with the primer, and it
was suggested that the amount of duplex formed between
the primer and the template may be constant, similar to a
transcription complex (<italic toggle="yes"><xref rid="bi026914ab00027" ref-type="bibr"></xref></italic>). Such a model where minimal
base-pairing occurs between the 3‘-end of the primer and
the template can more easily explain the modest observed
difference in affinity between 18TAG and 18TTA (∼3-fold)
as compared with the enormous difference expected (∼7.9
kcal mol<sup>-1</sup>) if the primer could form a complete 11-base-pair duplex with the template. On the basis of our results,
we favor a model that involves minimal base-pairing between
primer and template, and one where most of the energy of
stabilization comes from primer−protein contacts. At room
temperature, the 18-nt telomeric primers had <italic toggle="yes">k</italic><sub>off</sub> values
ranging from 0.047 to <0.00058 min<sup>-1</sup>, which translate into
Δ<italic toggle="yes">G</italic> values ranging from −12.4 to −15.0 kcal mol<sup>-1</sup> (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026914ab00028" ref-type="bibr"></xref>,
<xref rid="bi026914ab00029" ref-type="bibr"></xref></named-content></italic>). These values clearly dwarf the energy contributions
resulting from base-pairing. This was further illustrated when
the nontelomeric primers were examined. The NT18GGG
and NT18GTT primers had respective <italic toggle="yes">k</italic><sub>off</sub> values of 0.0048
and 0.40 min<sup>-1</sup>, which represent Δ<italic toggle="yes">G</italic> values of −13.7 and
−11.1 kcal mol<sup>-1</sup>. While the three 3‘-nucleotides may be
important for alignment of the primer with the template, the
contribution from these base-pairs toward stabilization of the
primer would be insignificant. Furthermore, we observed that
the telomerase−primer complex becomes more labile as the
ionic strength of the buffer increases, which again indicates
that protein−nucleic acid interactions are the predominant
force stabilizing the complex (G. Wallweber, unpublished
data); a more stable complex would be expected if the
majority of the stabilizing energy were the result of duplex
formation.
</p><p>Observations from a different experimental approach also
resulted in the similar conclusion: that the high affinity
between telomerase and 18GGG was primarily the result of
protein contacts. We compared the binding of this primer to
telomerase with that of the potent hTR template antagonist,
GRN163. Following binding to telomerase and subsequent
treatment with proteinase K, we found that GRN163
remained associated with hTR, while 18GGG binding was
lost (data not shown). While GRN163 formed a stable
interaction with hTR and 18GGG presumably with hTERT,
interestingly, their binding to telomerase was competitive
with each other, indicating an overlap of the binding sites.
</p><p>The variance in affinity with respect to the primer register
was observed with the nontelomeric primers as well as with
the telomeric series, which supported the idea of a significant
interaction between the enzyme and the 3‘-terminus of the
primer. There was at least an 80-fold difference between the
nontelomeric primer ending in GTT-3‘ (NT18GTT) versus
the one ending in GGG-3‘ (NT18GGG). The difference in
base-pairing (+1.1 kcal mol<sup>-1</sup> for a GTT DNA−RNA duplex
as compared with a GGG DNA−RNA duplex; ref <italic toggle="yes">28</italic>) once
again cannot explain the difference in stability that was
observed. The register ending in GGG-3‘ may be optimally
positioned in relation to the protein subunit to form a
stabilizing interaction. In this simplistic model, primers
ending in GGT-3‘, GTT-3‘, and TTA-3‘ would be respectively more distant and increasingly rotated away from a
given protein site and thus less able to form an interaction
there. The TTA-3‘ register is 3-nt removed from the putative
site of interaction and has the weakest affinity. The next
register, TAG-3‘, can potentially bind in two different
positions, the fully extended pretranslocation position or the
post-translocation position. Primer 18TAG had a monophasic
decay profile for its <italic toggle="yes">k</italic><sub>off</sub>, which we interpret as binding
primarily in a single position (Figure <xref rid="bi026914af00002"></xref>G). Furthermore, the
<italic toggle="yes">k</italic><sub>off</sub> of 18TAG determined using a competitive blocked primer
and pulse-labeling with [α-<sup>32</sup>P] dGTP were similar to the
<italic toggle="yes">k</italic><sub>off</sub> determined in Figure <xref rid="bi026914af00002"></xref> (data not shown); thus, we believe
that we have measured the affinity of 18TAG in the post-translocation position. On the basis of our model, we would
predict a primer bound in the pretranslocation position to
be the same or weaker than the primer ending in TTA-3‘.
From a teleological point of view, a weak interaction in this
register would be favorable to an incipient translocation
following the addition of the first G nucleotide. The post-
translocation register is 2-nt distance from the optimal register
(rotated in the opposite direction as compared to GTT-3‘)
and has a similar affinity to the primer ending in GTT-3‘.
</p><p>While it is difficult to directly compare the dissociation
rates between the human enzyme with that of the <italic toggle="yes">Euplotes</italic>
enzyme, as there were minor differences in the conditions
used, it appears that primers for the human enzyme (in certain
registers) are able to bind their cognate enzyme more tightly
(<italic toggle="yes"><xref rid="bi026914ab00027" ref-type="bibr"></xref></italic>). This distinction might be expected given the apparent
differences in processivity between the two enzymes. Interestingly, in <italic toggle="yes">Euplotes</italic> the <italic toggle="yes">k</italic><sub>off</sub> values for the different primers
seem to be invariant with respect to the primer's register
(<italic toggle="yes"><xref rid="bi026914ab00027" ref-type="bibr"></xref></italic>).
</p><p>We conducted all of our binding assays under identical
conditions with the exception of those that involved the
primers having a terminal 3‘-amino-G or 3‘-azido-G. For
these two primers, the 3‘-terminal nucleotide was added by
telomerase using 18AGG as the initial substrate together with
either 3‘-amino-dGTP or 3‘-azido-dGTP. Since the presence
of dGTP analogues was a modification in our binding
conditions, we examined their effect on the <italic toggle="yes">k</italic><sub>off</sub> of the 18GGG
primer. We observed an approximately 2-fold increase in
the <italic toggle="yes">k</italic><sub>off</sub> for that primer and subsequently found that ddGTP
as well as rGTP had a similar effect on 18GGG (unpublished
observation, R. Pruzan). These observations resulted in our
imputing a 2-fold increase in the <italic toggle="yes">k</italic><sub>off</sub> values for the primers
with 3‘-amino-dG and 3‘-azido-dG. We did not explore our
observations regarding the presence of nucleotides and their
affect on primer affinity further. While the ddGTP is a readily
accepted substrate that can be added to a primer, rGTP is
not (unpublished observation, R. Pruzan) and raises the
interesting question as to the possibility of a second nucleotide-binding site and whether nucleotide hydolysis may be
involved in this phenomenon. Interestingly, dGTP was
previously shown to affect the binding of certain primers to
the <italic toggle="yes">Euplotes</italic> enzyme, and it was postulated that dGTP may
provide a source of energy for translocation (<italic toggle="yes"><xref rid="bi026914ab00027" ref-type="bibr"></xref></italic>). Answering
these interesting questions and others such as the effect of
different nucleotides upon primers having different registers,
however, will require a separate study and is beyond the
scope of this work.
</p><p>In a separate but related observation, we found that the
<italic toggle="yes">k</italic><sub>off</sub> values for primers terminating with GGG-3‘, GTT-3‘,
and TTA-3‘ appeared to be the same whether their 3‘-ends
were generated by telomerase or were presented to the
enzyme as exogenously synthesized primers. For example,
starting with a primer ending with AGG-3‘ and allowing the
enzyme to extend this primer by a single nucleotide, using
[α-<sup>32</sup>P]-dGTP, resulted in a stable enzyme−primer interaction, similar to one ending in GGG-3‘ (unpublished
observations, G. Wallweber). Conversely, 18GGG, which
forms a very stable complex with the enzyme, could be
extended with dTTP and [α-<sup>32</sup>P] dATP, and that extended
primer ending in 3‘-dA was observed to have a similar <italic toggle="yes">k</italic><sub>off</sub>
as that of a primer ending in TTA-3‘ (unpublished observations, G. Wallweber). This suggests there is no difference
in primer recognition by an enzyme in an elongation mode
as compared to an enzyme in a static mode. These experiments, however, were carried out at nucleotide concentrations
well below the <italic toggle="yes">K</italic><sub>m</sub> of nucleotides and therefore are different
than the previously mentioned experiments with dGTP
analogues, which were conducted at concentrations of
nucleotide well above its <italic toggle="yes">K</italic><sub>m</sub>.
</p><p>While the 3‘-end of the primer was important, we also
observed a contribution to the enzyme−primer complex
stability from telomeric sequences at the 5‘-end of the primer,
as well as a limited contribution from base-pairing, and thus
are able to discern three separable regions of the primer that
can interact with telomerase. In addition to the previously
proposed template and anchor sites, we have identified an
interaction at the 3‘-end of the primer that can add 1−2 orders
of magnitude to its affinity depending upon its 3‘-register
with respect to hTR. We have further defined the nature of
this 3‘-interaction by appending the 2‘ and 3‘ positions of
the 3‘-ultimate sugar residue. No single modification affected
the affinity more than about 3−4-fold. A more systematic
study involving various 3‘-terminal nucleotides and the
enzyme may be required.
</p><p>Also, further studies including structural ones will be
required to map and understand all the interactions involved
between telomerase and its primer substrate. These, however,
must await the production of large amounts of active enzyme.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Dr. C. B. Harley for critically reading the
manuscript.
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Phone: (650) 473-8631. E-mail: rpruzan@geron.com.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2002-12-17</dateCreated><dateIssued encoding="w3cdtf">2003-01-21</dateIssued><copyrightDate encoding="w3cdtf">2003</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>Telomerase is a ribonucleoprotein responsible for maintaining the ends of linear chromosomes in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line and progenitor cell populations. In the absence of telomerase activity, telomeres shorten with each cell division until a critical length is reached, which can result in the cessation of cell division. The enzyme is required for cell immortality, and its activity has been detected in the vast majority of human tumors. Because of this, telomerase is an attractive target for inhibition in anticancer therapy. To learn more about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have used highly purified human enzyme and employed a two-primer method for determining the dissociation rates of these primers. Primers having sequence permutations of (TTAGGG)3 were found to have considerably different affinities. They had t1/2 values that ranged from 14 min to greater than 1200 min at room temperature. A primer ending in the GGG register formed the most stable complex with the enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold decrease in the koff. We have found that interactions of telomerase with primer substrates are stabilized mainly by contacts with the protein subunit of the enzyme (hTERT). 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