Role of the 70-kDa subunit of human replication protein A (I). Single-stranded dna binding activity, but not polymerase stimulatory activity, is required for DNA replication.
Replication protein A (RPA), also known as human single-stranded DNA-binding protein, is a three-subunit protein complex with multiple functions. Here, we investigated the role of the 70-kDa RPA subunit (p70) in DNA replication, by generating a series of deletion mutants. Mutant p70, which lacked 50 amino acids at the C-terminus, failed to interact with the 11-kDa RPA subunit (p11) and, when deleted further at the C terminus, was unable to interact with either the 34-kDa subunit (p34) or with p11, suggesting that p70 directly interacts with both p34 and p11. Studies with purified RPA mutants indicated that deletions at the N-terminal domain of p70 had very little effect on RPA's single-stranded DNA (ssDNA) binding activity, whereas deletion of amino acids 169-246 significantly weakened the DNA binding ability of RPA. By deleting amino acids 296-373 or 374-458, we totally abolished p70's ssDNA binding activity, suggesting that multiple p70 domains are involved in DNA binding. Two p70 domains, the N-terminal domain and the DNA binding domain, were required to stimulate DNA polymerase (pol) alpha, yet the DNA binding domain alone supported pol delta activity. Interestingly, RPA containing p70 with a zinc-finger domain deletion retained its DNA binding activity, but inhibited pol alpha and delta activity. RPA that lacked ssDNA binding activity failed to support simian virus 40 (SV40) DNA replication in vitro, whereas mutant RPA that lacked pol alpha stimulatory activity (including the zinc-finger p70 mutant) functioned normally. We conclude that RPA's DNA binding activity, but not its pol alpha stimulatory activity, is required for DNA replication.
Kim
D K
DK
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794, USA.
Stigger
E
E
Lee
S H
SH
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae; D001483:Base Sequence; D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III; D017931:DNA Primers; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051716:Replication Protein A; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase