Identification and characterization of the fourth single-stranded-DNA binding domain of replication protein A.
Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2. Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B. The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D). The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast. Interestingly, the amino-terminal portion of domain C contains a putative Cys4-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32. We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc. The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.
Brill
S J
SJ
Department of Molecular Biology, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855, USA. brill@mbcl.rutgers.edu
Bastin-Shanower
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002470:Cell Survival / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D051716:Replication Protein A; D014764:Viral Proteins; D015032:Zinc; D003545:Cysteine