Amino acids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro

Although p48 is the most conserved subunit of mammalian DNA polymerase a-pr imase (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 t he protein. Chimerical human-murine p48 was prepared and coexpressed with t hree wild-type subunits of pol-prim, and four subunit protein complexes wer e purified. All enzyme complexes synthesized DNA on single-stranded (ss) DN A and replicated simian virus 40 DNA. Although the recombinant protein comp lexes physically interacted with PyV T antigen (Tag), we determined that th e murine region A mediates the species specificity of PyV DNA replication i n 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 th at amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the funct ional cooperation of RPA, pol-prim, and PyV Tag.