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

Abstract

The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) ␣-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol ␣-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.Studies employing the simian virus 40 (SV40) in vitro replication system have been used to establish much of what is known about the enzymology of eukaryotic DNA replication (10,40,81). This system has also been used to study the reaction mechanisms that take place during particular stages in the replication process; for instance, during the initiation of DNA replication. SV40 replication is initiated when T antigen (T-ag), the single viral protein necessary for replication, site specifically binds to the viral origin (reviewed in references 8, 11, and 31). Upon binding, T-ag assembles into a double hexamer (21,23,51,61,77) that is able to function as a helicase (22,34,66,68,84). Owing to its helicase activity, T-ag is capable of catalyzing origin-specific unwinding, provided replication protein A (RPA) and topoisomerase I are present in the reaction (15,22,27,88).Recent insights into initiation of DNA replication at the SV40 origin include images of T-ag double hexamers assembled on the viral core origin (77). Additional studies have helped to define the minimal core origin sequences necessary for T-ag double hexamer formation (41,42,67). Further insights into T-ag's interactions with the central region of the core origin, site II, were provided by the solution structure of the T-ag origin binding domain (T-ag-obd 131-260 ) (49). These studies established that a pair of loops is utilized to make sequence-specific contacts with site II (49); a similar surface is used by the DNA-binding domain of papillomavirus to bind to DNA (29). Recent experiments have also helped to unravel how T-ag's interactions with the core origin are controlled by the cell cycle machinery (5; reference 83 and references therein).The SV40 replication system has also been used to investigate the formation of nascent DNA in the vicinity of the SV40 origin following T-ag cataly...

Analyses of the Interaction between the Origin Binding Domain from Simian Virus 40 T Antigen and Single-Stranded DNA Provide Insights into DNAUnwinding and Initiation of DNA Replication

“…These and related observations led to the hypothesis that T-ag, RPA, and the polymerase ␣-primase complex form a preinitiation complex (reviewed in references 10, 11, 21, 33, 66, and 84). Based on the current findings, it is possible that upon emergence of ssDNA out of the gap in the T-ag OBD hexamer, the polymerase ␣-primase complex initiates primer-RNA/ DNA synthesis at pyrimidine-rich trinucleotide sequences (12,13,50,51,57). Competition for RPA would then enable replication factor C to displace the polymerase ␣-primase complex and to load proliferating cell nuclear antigen (94).…”