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Nucleic Acids Res. 2015 Feb 27;43(4):2102-15. doi: 10.1093/nar/gkv095. Epub 2015 Feb 6.

Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing.

Author information

1
Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
2
Seattle Biomedical Research Institute, 307 Westlake Avenue, Seattle, WA 98109-5219, USA.
3
Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.
4
Seattle Biomedical Research Institute, 307 Westlake Avenue, Seattle, WA 98109-5219, USA Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, WA 98195, USA Department of Global Health, University of Washington, Seattle, WA 98195, USA.
5
Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands p.borst@nki.nl.

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.

PMID:
25662217
PMCID:
PMC4344527
DOI:
10.1093/nar/gkv095
[Indexed for MEDLINE]
Free PMC Article

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