Strand displacement synthesis by yeast DNA polymerase ε
نویسندگان
چکیده
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.
منابع مشابه
Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps
The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo(-) to carry out st...
متن کاملCMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.
DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synt...
متن کاملPif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase Î ́
Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase . We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5′–3′ hel...
متن کاملPif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ
Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5'-3' he...
متن کاملStrand displacement during deoxyribonucleic acid synthesis at single strand breaks.
Synthesis of DNA by DNA polymerases has been studied by using circular duplex DNA templates which contain single phosphodiester bond interruptions (nicks). Escherichia coli DNA polymerase can initiate synthesis at nicks by the covalent attachment of nucleotides to the 3’ end of the primer strand. An initial phase of synthesis is accompanied by the removal of nucleotides from the 5’ end of the n...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
دوره 44 شماره
صفحات -
تاریخ انتشار 2016