Lagging strand DNA synthesis by calf thymus DNA polymerases α, β, δ and ε in the presence of auxiliary proteins

Abstract

By using a defined gapped DNA substrate that mimicks a lagging strand of 230 nucleotides and that contains a defined pause site, we have analyzed calf thymus DNA polymerases (pol) α, β, δ, and ε in the presence of the three auxiliary proteins proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) for their ability to complete an Okazaki fragment. Pol α alone could fill the gap to near completion, but was strongly stopped by the pause site. Addition of low amounts of RP-A resulted in an increased synthesis by pol α past the pause site. In contrast, high amounts of RP-A strongly inhibited gap filling by pol α. Further inhibition was evident when the two other auxiliary proteins, PCNA and RF-C, were added in addition to RP-A. Pol β could completely fill the gap without specific pausing and also was strongly inhibited by RP-A. PCNA and RF-C had no detectable effect on pol β. Pol δ, relied as expected, on all three auxiliary proteins for complete gap filling synthesis and could, upon longer incubation, perform a limited amount of strand displacement synthesis. Pol ε core enzyme was able to fill the gap completely, but like pol α, essentially stopped at the pause site. This pausing could only be overcome upon addition of PCNA, RF-C and E.coli single-stranded DNA binding protein. Thus pol ε holoenzyme preferentially synthesized to the end of the gap without pausing. Ligation of the DNA products indicated that pol β core enzyme, pol δ and pol ε holoenzymes (but not pol α and pol ε core enzyme) synthesized products that were easily ligatable. Our results indicate that pol ε holoenzyme fills a defined lagging strand gapped template to exact completion and is able to pass a pause site. The data favour the hypothesis of Burgers (Burgers, P.M.J. (1991) J.Biol. Chem. 266, 22698-22706) that pol ε might be a candidate for the second replication enzyme at the lagging strand of the replication fork. © 1993 Oxford University Press.