Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing.
Identifieur interne : 000F68 ( Main/Corpus ); précédent : 000F67; suivant : 000F69Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing.
Auteurs : Paul-Andre Genest ; Loren Baugh ; Alex Taipale ; Wanqi Zhao ; Sabrina Jan ; Henri G A M. Van Luenen ; Jonas Korlach ; Tyson Clark ; Khai Luong ; Matthew Boitano ; Steve Turner ; Peter J. Myler ; Piet BorstSource :
- Nucleic acids research [ 1362-4962 ] ; 2015.
English descriptors
- KwdEn :
- MESH :
- chemical , analogs & derivatives : Uracil.
- chemical , analysis : Glucosides, Uracil.
- chemical , metabolism : DNA-Binding Proteins, Glucosides, Protozoan Proteins, Uracil.
- genetics : Leishmania, Plasmids.
- Sequence Analysis, DNA.
Abstract
Base J (β-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.
DOI: 10.1093/nar/gkv095
PubMed: 25662217
PubMed Central: PMC4344527
Links to Exploration step
pubmed:25662217Le document en format XML
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<term>Glucosides (metabolism)</term>
<term>Leishmania (genetics)</term>
<term>Plasmids (genetics)</term>
<term>Protozoan Proteins (metabolism)</term>
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<front><div type="abstract" xml:lang="en">Base J (β-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication. </div>
</front>
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<Abstract><AbstractText>Base J (β-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication. </AbstractText>
<CopyrightInformation>© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.</CopyrightInformation>
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