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 alpha-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.
Kautz
A R
AR
Abteilung Biochemie, Institut für Molekulare Biotechnologie e.V., D-07745 Jena, Germany.
Weisshart
K
K
Schneider
A
A
Grosse
F
F
Nasheuer
H P
HP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010649:Phenylalanine; D011120:Polyomavirus / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051716:Replication Protein A; D012694:Serine; D013539:Simian virus 40 / Q000235:genetics; D013045:Species Specificity; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; D012694:Serine; D010649:Phenylalanine; D004247:DNA; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I