Strand displacement synthesis by yeast DNA polymerase ε.
DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair.
Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden Howard Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NY 10016, USA.
Rais A
RA
Ganai
Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616-8665, USA.
Xiao-Ping
XP
Zhang
Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616-8665, USA.
Wolf-Dietrich
WD
Heyer
Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden erik.tm.johansson@umu.se.
Erik
E
Johansson
D004257:DNA Polymerase II / Q000378:metabolism*; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects
D004271:DNA, Fungal; D009711:Nucleotides; D021122:Protein Subunits; D012965:Sodium Chloride; D004257:DNA Polymerase II