Sloppy bypass of an abasic lesion catalyzed by a Y-family DNA polymerase.
DNA damage that eludes cellular repair pathways can arrest the replication machinery and stall the cell cycle. However, this damage can be bypassed by the Y-family DNA polymerases. Here, Dpo4, an archetypal Y-family member from the thermophilic Sulfolobus solfataricus, was used to extend our kinetic studies of the bypass of an abasic site, one of the most mutagenic and ubiquitous cellular lesions. A short oligonucleotide sequencing assay is developed to directly sequence DNA bypass products synthesized by Dpo4. Our results show that incorporation upstream of the abasic lesion is replicated error-free; yet dramatically, once Dpo4 encounters the lesion, synthesis became sloppy, with bypass products containing a myriad of mutagenic events. Incorporation of dAMP (29%) and dCMP (53%) opposite the abasic lesion at 37 degrees C correlates exceptionally well with our kinetic results and demonstrates two dominant bypass pathways via the A-rule and the lesion loop-out mechanism. Interestingly, the percentage of overall frameshift mutations increased from 71 (37 degrees C) to 87% (75 degrees C). Further analysis indicates that lesion bypass via the A-rule is strongly preferred over the lesion loop-out mechanism at higher temperatures and concomitantly reduces the occurrence of "-1 deletion" mutations observed opposite the lesion at lower temperatures. The bypass percentage via the latter pathway is confirmed by an enzymatic digestion assay, verifying the reliability of our sequencing assay. Our results demonstrate that an abasic lesion causes Dpo4 and possibly all Y-family members to switch from a normal to a very mutagenic mode of replication.
Fiala
Kevin A
KA
Department of Biochemistry, the Ohio State Biochemistry Program, the Comprehensive Cancer Center, Ohio State University, Columbus 43210, USA.
Suo
Zucai
Z
D002384:Catalysis; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008957:Models, Genetic; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics*
D029968:Escherichia coli Proteins; D009711:Nucleotides; C509838:Dpo4 protein, E coli; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase