Binding properties of replication protein A from human and yeast cells.
Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.
Kim
C
C
Department of Biochemistry, University of Iowa, Iowa City 52242.
Snyder
R O
RO
Wold
M S
MS
D001483:Base Sequence; D001667:Binding, Competitive; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011131:Polyribonucleotides / Q000378:metabolism; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013539:Simian virus 40 / Q000378:metabolism
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011131:Polyribonucleotides; C497167:RPA1 protein, human; D051716:Replication Protein A; C027903:oligo (dT)