Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

Abstract

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase 8 that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase 8 on a DNA singly primed 4X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase 6, in contrast to DNA polymerase a, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase a and 8 are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.The in vitro origin of replication-dependent simian virus 40 (SV40) DNA replication has been established by using cytosolic extracts of human or monkey cells supplemented with SV40 large tumor antigen (Tag) (1-3). SV40 Tag is a multifunctional protein possessing origin-specific DNA-binding, ATPase, and DNA helicase activities; this antigen is required for initiating DNA replication (4-7).We have described a system capable of catalyzing a Tag and SV40 origin-dependent replication reaction using purified proteins. In addition to Tag and DNA containing the SV40 origin of replication (ori'DNA), immunopurified DNA polymerase a-DNA primase complex, topoisomerase (topo) I and/or II and a multisubunit single-stranded DNA-binding protein (SSB), all isolated from HeLa cells, are required (8,9). Replication reactions containing these proteins will hereafter be identified as the monopolymerase system.Proliferating-cell nuclear antigen (PCNA), a known accessory factor for DNA polymerase 6 (Pol-6) (10-12), was shown essential for leading-strand DNA synthesis in crude systems; without PCNA only small-sized DNA products arising from the lagging strand were synthesized (13). In contrast, PCNA had no effect on the monopolymerase system. The reasons for this difference were traced to the effect of multiple protein factors [an elongation inhibitor (El), activators I (Al) and II (All)], which were present in crude fractions but not in the monopolymerase system (14, 15). The El has been characterized as a 120-kDa protein that binds to ends of DNA chains, thus inhibiting enzymes that act at such sites (such as exonuclease III, 5' --3' exonuclease, and DNA ligase).Pol-6 was initially isolated and distinguished from other DNA polymerases by its tightly associated 3' -k 5' exonuclease activity and its PCNA dependency (16,17). Recently another polymerase, also called Pol-6, which does not require PCNA, has been isolated from several mammalian sources (17-20). These different Pol-8 preparations share some common properties, including 3' -+ 5' exonuclea...

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

“…Moreover, mutations in POL2 or DPB2 restore silencing at a mutant HMR silencer allele (71) and cause a loss of silencing at a wild type HMR allele. 2 By replicating heterochromatin, pol ⑀ would be ideally localized to facilitate and maintain the silencing of these heterochromatic genes.…”

“…pol ␣ is clearly involved in the synthesis and extension of RNA primers during DNA replication initiation (reviewed in Ref. 1), whereas pol ␦ is responsible for elongation, as most clearly demonstrated in SV40 DNA replication (2,3). Although pol ⑀ is not required for SV40 replication (4,5), it does appear to have a role in chromosomal DNA replication (5)(6)(7)(8)(9)(10).…”

Human Xeroderma Pigmentosum Group A Protein Interacts with Human Replication Protein A and Inhibits DNA Replication

“…In Vitro SV40 DNA Replication-The reactions were carried out as described previously (28). In brief, reaction mixtures (40 l) contained 40 mM creatine phosphate/di-Tris salt (pH 7.7), 1 g of creatine kinase, 7 mM MgCl 2 , 0.5 mM DTT, 4 mM ATP, 200 M UTP, GTP, and CTP, 100 M dTTP, dGTP, and dCTP, 20 M [␣-…”

Comparison of DNA Polymerases .ALPHA., .DELTA., and .EPSILON. of Mouse Cell Line FM3A and Its Temperature-Sensitive Mutant tsFT20.

“…On the other hand, the low activity of pol a in tsFT20 cells did not affect the cellular level of pol s activity. This was unexpected considering its proposed function as a leading strand polymerise during in vitro SV40 DNA replication (Prelich and Stillman 1988; Lee et al 1989;Weinberg and Kelly 1989). The intracellular activity of pol S may be controlled indirectly by regulation of its accessory factors, PCNA and the replication factor-C (the activator I) (Tsurimoto and Stillman 1990; , although this possibility has not been studied extensively.…”

“…The contribution of poi s to DNA replication has been suggested by the dependence of its DNA synthesis activity and processivity on the proliferating cell nuclear antigen (PCNA) (Tan et al 1986; Prelich et al 1987a), which is expressed at an increased level in cells stimulated to proliferate (Celis et al 1987). In addition, pol S was found to be necessary as a leading strand DNA polymerase in the in vitro SV40 DNA replication system (Prelich and Stillman 1988; Lee et al 1989;Weinberg and Kelly 1989), where PCNA is also required as one of the accessory proteins of pol 6 (Prelich et al 1987b). The much higher processivity of pol S in the presence of PCNA than that of poi a supports the idea that pol 6 synthesizes the leading strand at the replication fork.…”