Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

Abstract

Although p48 is the most conserved subunit of mammalian DNA polymerase ␣-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein.Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.The small DNA tumor viruses simian virus 40 (SV40) and mouse polyomavirus (PyV) have provided excellent model systems to study the mechanisms and regulation of eukaryotic DNA replication (reviewed in references 7, 9, 55, and 67). Their DNA replication in vivo and in vitro largely depends on the replication apparatus of their hosts, as only one protein, namely, the multifunctional viral large T antigen (Tag), is supplied by the virus (20,26,49).The establishment of cell-free SV40 and PyV DNA replication systems was a major step towards the purification and characterization of essential host replication factors (67). These studies allowed the understanding of the molecular mechanisms involved in the assembly and progression of replication forks (7,28,67). Along with genetic and other biochemical approaches, these DNA replication systems provided insights into the processes of initiation and elongation (67). Interestingly, several parallels between eukaryotic and prokaryotic DNA duplication emerged which show that central processes of DNA metabolism are in part conserved in all kingdoms (33,60,61). The studies of the polyomaviral systems have allowed the development of a general model for eukaryotic DNA replication. In an initial step, Tag recognizes the replication origin (ori) and forms a double hexameric complex, which causes specific distortions of the double-stranded (ds) DNA (4,15,20,37,49). Subsequently, dsDNA is unwound, and multiprotein complexes are assembled by Tag and host replication proteins, such as eukaryotic single-stranded (ss) DNA-binding protein (replication protein A [RPA]), topoisomerase I (topo I), and DNA polymerase ␣-primase (polprim), are recruited (10,11,17,23,38,53,54). After binding and distortion of the dsDNA, the helicase function of Tag melts and unwinds the ds ori DNA (4, 59). For this function, Tag depends on RPA as a cofactor to stabilize the ssDNA resulting from the helicase activity of Tag (7,29,72). Th...

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

“…It appears therefore that multiple functions contribute to the species-specific requirement for human p180 and that the sequences involved are spread over a large part of the p180 protein. This situation differs from that found in polyomavirus DNA replication, where a short defined stretch of Mp48 controls the species specificity, and biochemical data in conjunction with protein structure predictions gave some insight into possible functions for this region (30). With p180 the situation is more complicated, and therefore detailed conclusions concerning the mechanism underlying species specificity will very likely require resolution of the three-dimensional structure of the initiation complex.…”

“…Although this system is not strictly species-specific, and the mechanism behind stimulation of primer synthesis by TAg is not entirely understood, it requires specific interactions of TAg with multiple sites on several or all RPA and DNA polymerase ␣-primase subunits (27,30,53). Substitution of Mp180 for Hp180 prevents stimulation by SV40 TAg.…”

“…In the case of PyV, further resolution was achieved with studies of DNA polymerase ␣-primase complexes containing chimeric p48 subunits consisting of various combinations of human and murine sequences. It was concluded that a lesser conserved stretch of amino acids (residues 257-288) from the murine subunit was necessary for successful PyV DNA replication, and it is likely that further study of the function of this region will shed light on the mechanism involved in this phenomenon (30,31). In this study we applied a similar approach in an attempt to determine which domains of human p180 are essential for initiation of SV40 DNA replication.…”

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

“…It is known that SV40 TAg independently interacts with all four subunits of Pol-prim and that these interactions are important for DNA replication and are involved in regulation of the species specificity of SV40 DNA replication (8,24,50,57,58). To systematically test the role of the individual subunits of Pol-prim in more detail, we expressed and purified different human-mouse chimeric Pol-prim complexes and studied their activities in the monopolymerase replication system.…”

Viral Interference with DNA Repair by Targeting of the Single-Stranded DNA Binding Protein RPA

“…Broadly, these can be divided into issues related to DNA replication or to control of cell phenotype. In productive infection, LT initiates viral DNA replication [18], has helicase [19] and ATPase activities [20] and associates with pol α-primase [21], as well as promotes integration of the viral genome into the host [22] or promotes recombination [23]. It has numerous effects on cell phenotype, many of which are dependent on its association with members of the retinoblastoma tumor suppressor family.…”