title: The choice of nucleotide inserted opposite abasic sites formed within chromosomal DNA reveals the polymerase activities participating in translesion DNA synthesis.
abstract: Abasic sites in genomic DNA can be a significant source of mutagenesis in biological systems, including human cancers. Such mutagenesis requires translesion DNA synthesis (TLS) bypass of the abasic site by specialized DNA polymerases. The abasic site bypass specificity of TLS proteins had been studied by multiple means in vivo and in vitro, although the generality of the conclusions reached have been uncertain. Here, we introduce a set of yeast reporter strains for investigating the in vivo specificity of abasic site bypass at numerous random positions within chromosomal DNA. When shifted to 37°C, these strains underwent telomere uncapping and resection that exposed reporter genes within a long 3' ssDNA overhang. Human APOBEC3G cytosine deaminase was expressed to create uracils in ssDNA, which were excised by uracil-DNA N-glycosylase. During repair synthesis, error-prone TLS bypassed the resulting abasic sites. Because of APOBEC3G's strict motif specificity and the restriction of abasic site formation to only one DNA strand, this system provides complete information about the location of abasic sites that led to mutations. We recapitulated previous findings on the roles of REV1 and REV3. Further, we found that sequence context can strongly influence the relative frequency of A or C insertion. We also found that deletion of Pol32, a non-essential common subunit of Pols δ and ζ, resulted in residual low-frequency C insertion dependent on Rev1 catalysis. We summarize our results in a detailed model of the interplay between TLS components leading to error-prone bypass of abasic sites. Our results underscore the utility of this system for studying TLS bypass of many types of lesions within genomic DNA.
authors.affiliation: Chromosome Stability Section, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA. Electronic address: chank2@niehs.nih.gov.
authors.forename: Kin
authors.initials: K
authors.lastname: Chan
authors.forename: Michael A
authors.initials: MA
authors.lastname: Resnick
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Gordenin
mesh_terms: D000071480:APOBEC-3G Deaminase; D015825:Chromosomes, Fungal; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D016254:Mutagenesis, Insertional; D009711:Nucleotides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D009711:Nucleotides; D029701:Saccharomyces cerevisiae Proteins; D014498:Uracil; D004259:DNA-Directed DNA Polymerase; D051981:Uracil-DNA Glycosidase; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase
keywords: APOBEC; Abasic site; Deoxycytidyltransferase; Single-strand DNA; Translesion DNA synthesis