Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

Abstract

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 strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.

Strand displacement synthesis by yeast DNA polymerase ε

“…It was previously shown that high concentrations of salt inhibit strand displacement synthesis by Pol δ exo − ( 28 ). To ask whether the salt concentration also influences strand displacement synthesis by Pol ε, we performed assays in the presence of various concentrations of NaAc (Figure 2 ).…”

“…We designed substrates containing flaps with a length of 1, 5, 10 or 20 nucleotides. It was recently shown that strand displacement synthesis by Pol δ exo − (pol3-5DV) is strongly inhibited when the blocking primer contains longer flaps ( 28 ). In contrast to Pol δ exo − , our results show that Pol ε exo − is not hindered by the flaps and is able to synthesize DNA to the end of the template by displacing the entire blocking oligo irrespective of the flap size (Figure 4D ).…”

Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ

“…DNA polymerase δ wild-type and D520V (Pol δ DV ), purified as previously described ( 27 – 29 ). Replication Protein A (RPA), PCNA and Replication Factor C (RFC) were purified from E. coli overproduction strains as described ( 19 , 30 , 31 ).…”

Dna2 processes behind the fork long ssDNA flaps generated by Pif1 and replication-dependent strand displacement

“…Importantly, rad27 rnh35 mutant cells do not support viability, suggesting that Fen1 and RNAase H2 have redundant roles in Okazaki fragment processing 6 . The Polδ- dependent strand displacement of the previous Okazaki fragment, which is stimulated by proliferating cell nuclear antigen (PCNA), is counterbalanced by its 3’–5’nucleolitic proof reading activity that resects the growing 3’ end of the Okazaki fragment 2 , 4 , 7 . Mutations in the proof reading activity of DNA polymerase δ are lethal in combination with RAD27 deletion, suggesting that DNA polymerase δ-dependent degradation of the growing 3’ ends of the Okazaki fragments and Fen1 synergize in the maturation of the Okazaki fragments 8 .…”