Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Abstract

Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase ␣-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase ␣-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase ␣-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase ␣ depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase ␣-primase but rather on sequences in the C-terminal region of human p180.Polyomavirus DNA replication has been studied extensively because of the ease with which viruses of this family, which include SV40 and mouse polyoma virus (PyV), 1 can be grown in cell culture and moreover because of the availability of an in vitro replication system, which allows detailed investigation of the individual factors that play a role in the replication process (1, 2). Polyomavirus DNA replication has been found to be largely dependent upon factors involved in replication of the host DNA but does contribute one essential trans-acting factor known as large T antigen (TAg) to the replication complex. TAg is responsible for recognition of the viral origin of replication, at which it forms a double hexamer (3). In the presence of the single-stranded DNA-binding protein, RPA (replication protein A), and topoisomerase I, TAg proceeds to unwind the origin DNA and recruits the DNA polymerase ␣-primase heterotetramer, which then initiates bidirectional DNA synthesis (4 -11). DNA polymerase ␣-primase initiates DNA replication through the action of its smallest subunit, p48, which synthesizes RNA primers that are subsequently elongated by the large subunit, p180 (12-15). The bulk of DNA synthesis is, however, carried out by a more processive enzyme complex consisting of DNA polymerase ␦ and its processivity factor, PCNA (16 -18). The transition between DNA synthesis by DNA polymerases ␣ and ␦ is mediated by the PCNA loading factor replication factor C (16, 19). Throughout the viral replication cycle, TAg double hexamers are thought to act as the replicative helicase (20). For a more extensive account of the DNA replication process and its participating factors see Refs. 1, 2, and 21-23. SV40 and PyV are very similar with regard to DNA replication; their respectiv...

Restriction of Human Polyomavirus BK Virus DNA Replication in Murine Cells and Extracts

“…In contrast, the addition of hPol ␣-primase resulted in SV40 TAg-dependent replication of SV40 origin-containing DNAs to about 60% of the level obtained with mPyV TAg and its cognate origin (Fig. 4, compare bars 7 and 1), consistent with published data (90). Similarly, purified human recombinant RPA (hRPA) added to murine extracts did not support replication of BKV DNA (Fig.…”

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

“…SV40 TAg has been shown to interact physically with purified Pol-prim and independently with all four Polprim subunits (11,46,57). In contrast, physical interactions of purified Pol-prim with BKV TAg have not been investigated.…”

“…Initiation reactions were performed as previously described (29,31,46) Recombinant Pol-prim was added as indicated in the figure legends. Half of the reaction mixture was spotted on DE 81 paper (Whatman), precipitated with cold 10% (wt/vol) TCA containing 2.5% (wt/vol) sodium pyrophosphate, washed with 1 M HCl, and analyzed by scintillation counting.…”

“…on May 9, 2018 by guest http://jvi.asm.org/ in human and mouse cell extracts, respectively (8,23,46,50). The activity of human and mouse Pol-prim was tested in assays with ssDNA as a template ( Fig.…”

8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells

“…1) that is replication incompetent (23). Transfections lacking DNA were done to assess the background level of phospho-ERK (P-ERK) in the absence of Ras overexpression; normal and mutant constructs were also used as controls to provide the lower and upper limits of the P-ERK signal.…”