Primer-DNA formation during simian virus 40 DNA replication in vitro.

Abstract

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (-30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase a-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species.Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.With the exception of a single virally encoded protein termed T antigen (T-ag), replication of simian virus 40 (SV40) is dependent on simian or human host proteins (47). The dependence of SV40 replication on host proteins, and the presence of a single, well-defined origin of replication, has made SV40 a useful model system for studies of DNA replication in higher eukaryotes.An in vitro SV40 replication system was developed by Li and Kelly (28), using simian extracts, and extended to human extracts by Stillman and Gluzman (44) and Wobbe et al. (54). This system has enabled the identity and function(s) of many of the host proteins required for SV40 DNA replication to be established (for reviews, see references 8, 22, and 43). It has also permitted an analysis of the mechanisms operating during various stages of the SV40 DNA replication to be studied in detail. We have used this system to study the T-ag-dependent initiation of SV40 DNA synthesis (4, 5).The role played by T-ag during initiation of SV40 DNA replication has been reviewed recently (la, 18). In summary, T-ag recognizes GAGGC binding sites within the SV40 origin, assembles as a double hexamer (30), and melts the early palindrome within the SV40 origin (2). In the presence of topoisomerase I and a single-stranded binding protein, such as the three-subunit complex present in humans (HSSB, also termed RFA or RPA) (8,22,43), the inherent helicase activity of T-ag (42) catalyzes further unwinding of the origin region (11,12,55). T-ag-dependent unwinding of the SV40 origin is a critical initiation event, since it allows additional replication factors to gain entry to the origin region. Less is known about subsequent steps during initiation of SV40 DNA replication.We have conducted pulse-labeling experiments designed to more fully characterize postunwinding initiation events.

The Trans-autostimulatory Activity of Rad27 Suppresses dna2 Defects in Okazaki Fragment Processing

“…7, A and B, lanes 13-15). Collectively, these results indicate that Rad27(367-382) stimu- 8 and 14 -16) and GST-Rad27 (lanes 9 -11 and 17-19) with Dna2 (0.05 fmol; lanes [5][6][7][8][9][10][11][12] or Dna2⌬405N (0.05 fmol; lanes [13][14][15][16][17][18][19][20]. B, overexpression of Rad27 mutant proteins under control of the ADH1 promoter in the dna2⌬405N strain suppressed the ts growth defects.…”

“…In DNA replication, lagging strand DNA synthesis is more complex than leading strand because it requires discontinuous DNA synthesis and joining of short pieces of DNA called Okazaki fragments (8,9). In brief, polymerase (pol) 2 ␣-primase synthesizes an initial RNA-DNA primer on the lagging strand template that is extended by pol ␦ to generate a new Okazaki fragment.…”

The Evolutionarily Conserved Zinc Finger Motif in the Largest Subunit of Human Replication Protein A Is Required for DNA Replication and Mismatch Repair but Not for Nucleotide Excision Repair

“…Pulse Labeling for Early DNA Product-The assay is essentially based on previous reports (52,53). Reaction mixtures (50 l) contained 7 mM MgCl 2 , 0.5 mM DTT, 4 mM ATP, 40 mM creatine phosphate, 30 mM HEPES (pH 7.8), 0.2 units of creatine kinase, 0.6 g of supercoiled SV40 origin-containing plasmid (pSV010), 0.84 g of T Ag, 1.4 g of recombinant RPA preparations as indicated, and RPA-depleted 293 cell lysate.…”

“…1A are synthesized during the elongation phase of a 60-min reaction. If DNA synthesis is initiated (but the products are not significantly elongated) one expects to detect the initial Okazaki fragments in a short pulse-labeled SV40 DNA replication reaction (53).…”

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

“…It was previously demonstrated that the Pol ␣-primase complex initiates DNA replication by synthesizing a small RNA/DNA hybrid that has been termed primer-RNA/DNA (16,(56)(57)(58). Primer-RNA/DNA is formed exclusively from lagging-strand DNA templates, initially in the vicinity of the SV40 origin and, at later stages of synthesis, at progressively distal locations (13,25,50). Once synthesized, primer-RNA/DNA molecules are extended by a second PCNA-dependent polymerase to form full-length Okazaki fragments (16,(56)(57)(58)78).…”