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.
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