title: Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing.
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.
authors: Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors: Paul-Andre
authors: PA
authors: Genest
authors: Seattle Biomedical Research Institute, 307 Westlake Avenue, Seattle, WA 98109-5219, USA.
authors: Loren
authors: L
authors: Baugh
authors: Seattle Biomedical Research Institute, 307 Westlake Avenue, Seattle, WA 98109-5219, USA.
authors: Alex
authors: A
authors: Taipale
authors: Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors: Wanqi
authors: W
authors: Zhao
authors: Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors: Sabrina
authors: S
authors: Jan
authors: Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors: Henri G A M
authors: HG
authors: van Luenen
authors: Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.
authors: Jonas
authors: J
authors: Korlach
authors: Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.
authors: Tyson
authors: T
authors: Clark
authors: Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.
authors: Khai
authors: K
authors: Luong
authors: Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.
authors: Matthew
authors: M
authors: Boitano
authors: Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.
authors: Steve
authors: S
authors: Turner
authors: 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.
authors: Peter J
authors: PJ
authors: Myler
authors: Division of Molecular Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands p.borst@nki.nl.
authors: Piet
authors: P
authors: Borst
mesh_terms: D004268:DNA-Binding Proteins / Q000378:metabolism; D005960:Glucosides / Q000032:analysis* / Q000378:metabolism; D007891:Leishmania / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015800:Protozoan Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014498:Uracil / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D005960:Glucosides; C402790:J-specific DNA-binding protein, protozoa; D015800:Protozoan Proteins; C084880:5-((glucopyranosyloxy)methyl)uracil; D014498:Uracil