Initiation of simian virus 40 DNA synthesis in vitro.

Abstract

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of liitiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.With the exception of a single virus-encoded protein, the simian virus large tumor antigen (T-Ag), replication of simian virus 40 (SV40) DNA is dependent on host proteins from permissive simian or human cells (57). SV40 in vitro replication systems have been developed that require T-Ag, plasmids containing the SV40 origin, an ATP-regenerating system, and extracts prepared from either simian or human cells (30,51,66). Studies with the SV40 in vitro replication systems have enabled the identification of host proteins involved in SV40 and presumably chromosomal DNA replication (reviewed in references 7 and 49). As a result of these studies, the polymerase a (pol a)-primase complex was shown to be essential for SV40 DNA replication (30,36) and to play a role in establishing the host range of SV40 DNA replication (36). Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA pol 8 have been also demonstrated to be required for SV40 replication (26,43,70), especially for synthesis of leading strands (44). In addition, efficient elongation of newly synthesized DNA is known to require a factor termed activator 1. An inhibitor of DNA synthesis, poly(ADP-ribose) polymerase, copurifies with activator 1 through several isolation steps, but the two activities can be separated, and whether poly(ADP-ribose) polymerase plays a direct role in DNA synthesis is unknown (28,29). A protein similar in activities and subunit structure to activator 1 has been isolated and termed RF-C (62). To...

Replication terminus for DNA polymerase I during initiation of pAMβ1 replication: role of the plasmid‐encoded resolution system

“…Interestingly, a programmed switch between an initiating (Pol␣) and an elongating (Pol␦) polymerase might also take place in vivo during SV40 replication. Indeed, while the switch between the two polymerases occurs in vitro 200-300 nt from the SV40 origin, in crude extracts, the switch might occur earlier as Pol␣ progresses for only 30 nt (Bullock et al, 1991;Tsurimoto and Stillman, 1991;Waga and Stillman, 1994). Another possible case where such a programmed switch might occur is during replication of mammalian mitochondrial DNA.…”

The replicative polymerases PolC and DnaE are required for theta replication of the Bacillus subtilis plasmid pBS72

“…Based on systems for which a Pol III holoenzyme has been isolated and/or reconstructed in vitro, it is generally thought that both DNA strands of genomes are polymerized by a single replicative polymerase of the C (prokaryotes) or B (eukaryotes) families, dimerized in a holoenzyme structure (for reviews, see Benkovic et al, 2001;Bullock, 1997 , 2001;Lemon & Grossman, 1998, 2000. In the replication fork of the B. subtilis plasmid pAMb1, PolC might be mainly involved in leading strand synthesis while DnaE might be required for efficient lagging strand polymerization (Dervyn et al, 2001).…”

“…Based on systems for which a Pol III holoenzyme has been isolated and/or reconstructed in vitro, it is generally thought that both DNA strands of genomes are polymerized by a single replicative polymerase of the C (prokaryotes) or B (eukaryotes) families, dimerized in a holoenzyme structure (for reviews, see Benkovic et al, 2001;Bullock, 1997). However, this scheme might not be universal as replication in numerous organisms depends on two different polymerases.…”

“…Differences have also been documented in the DNA polymerase III (Pol III) holoenzyme, a subassembly of the replication machinery where DNA synthesis is catalysed. In E. coli this element contains 10 different subunits of which four (h, c, x and y) are missing in B. subtilis (Bruck & O'Donnell, 2000;Bruck et al, 2002;Lemon et al, 2002; MartinezJimenez et al, 2002;McHenry, 2003).Based on systems for which a Pol III holoenzyme has been isolated and/or reconstructed in vitro, it is generally thought that both DNA strands of genomes are polymerized by a single replicative polymerase of the C (prokaryotes) or B (eukaryotes) families, dimerized in a holoenzyme structure (for reviews, see Benkovic et al, 2001;Bullock, 1997 , 2001;Lemon & Grossman, 1998, 2000. In the replication fork of the B. subtilis plasmid pAMb1, PolC might be mainly involved in leading strand synthesis while DnaE might be required for efficient lagging strand polymerization (Dervyn et al, 2001).…”

On the presence of DNA polymerase α in human lymphocyte nuclei and chromosomes

“…Two structurally distinct types of DNA polymerases are known: (i) the alpha-type which includes polymerases , and , which are possibly involved in DNA synthesis of both leading and lagging strands; and (ii) the /3-type which includes the polymerases mostly involved in DNA repair mechanisms (Chiu & Ban!, 1975), although their possible role in DNA synthesis has only recently been suggested . Among the above cited enzymes, DNA polymerase has been studied in detail and appears to play a critical role in DNA synthesis because of its enhanced activity in *Correspondence Istituto di Biologia Generale, Facoltà di Medicina,Università,Via Ospedale 119,09124 Cagliari,Italy. proliferating cells (Bambara & Jessee, 1991), as well as its association with primase activity (Nethanel & Kaufmann, 1990;Bullock et al, 1991). DNA polymerase c exists in mammals as a complex with DNA primase and is composed of four subunits with a molecular mass of 180, 68-77, 55 and 48 kDa (Kozu et al, 1990).…”