A hierarchy of SSB protomers in replication protein A.
Replication Protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (SSB) found in all eukaryotic cells. RPA is known to be required for many of the same reactions catalyzed by the homotetrameric SSB of bacteria, but its origin, subunit functions, and mechanism of binding remain a mystery. Here we show that the three subunits of yeast RPA contain a total of four domains with weak sequence similarity to the Escherichia coli SSB protomer. We refer to these four regions as potential ssDNA-binding domains (SBDs). The p69 subunit, which is known to bind ssDNA on its own, contains two SBDs that together confer stable binding to ssDNA. The p36 and p13 subunits each contain a single SBD that does not bind stably, but corresponds to the minimal region required for viability in yeast. Photocross-linking of recombinant protein to ssDNA indicates that an SBD consists of approximately 120 amino acids with two centrally located aromatic residues. Mutation of these aromatic residues inactivates ssDNA binding and is a lethal event in three of the four domains. Finally, we present evidence that the p36 subunit binds ssDNA, as part of the RPA complex, in a salt-dependent reaction similar to the wrapping of ssDNA about E. coli SSB. The results are consistent with the notion that RPA arose by duplication of an ancestral SSB gene and that tetrameric ssDNA-binding domains and higher order binding are essential features of cellular SSBs.
Philipova
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D
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855, USA.
Mullen
J R
JR
Maniar
H S
HS
Lu
J
J
Gu
C
C
Brill
S J
SJ
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011995:Recombination, Genetic; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D051716:Replication Protein A