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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.
Highlights
During lagging strand DNA replication DNA polymerase ␦ (Pol ␦) performs three essential and basic functions in the process
During Okazaki fragment maturation Pol ␦ catalyzes strand displacement DNA synthesis through the downstream Okazaki fragment to allow for the generation of 5 -flaps that are substrates for the FEN1 endonuclease [1,2,3,6,7]
We used 3 –5 exonuclease deficient versions of Pol ␦ in order to determine the strand displacement synthesis activity of the polymerase without the possibility of subsequent reversal of strand displacement by its exonuclease activity [18]. This allowed us to focus on the basic biochemical properties of this enzymatic activity and ask how it is affected by the presence of 5 -flaps of different lengths in the DNA strand to be displaced, and how the presence of the single-stranded DNA binding protein Replication Protein A (RPA) affects the activity, and, how binding to proliferating cell nuclear antigen (PCNA) stimulates strand displacement. This approach allowed us to discover a novel property of Pol ␦, which we propose is regulated by PCNA
Summary
During lagging strand DNA replication DNA polymerase ␦ (Pol ␦) performs three essential and basic functions in the process. Via DNA-directed DNA synthesis Pol ␦ catalyzes extension of the short Okazaki fragments generated by DNA Pol ␣, thereby filling the gap between two successive Okazaki fragments [1,2,3] During this process Pol ␦ proofreads for mis-incorporated bases via its 3 –5 exonuclease activity, allowing for a relative high fidelity in copying the template strand [4,5]. Strand displacement by Pol ␦ and FEN1 cleavage activity must be a highly coordinated process to generate ligatable nicks that are the substrate of DNA ligase I (nick translation) [8] In this process the amount of strand displacement activity needs to be regulated to avoid generating 5 -flaps that are long enough to bind Replication Protein A (RPA), as RPA binding is inhibitory to FEN1 cleavage [9].
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