Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase alpha by multiple mechanisms.
Replication Protein A (RPA) from human cells is a stable complex of 70-, 32-, and 14-kDa subunits that is required for multiple processes in DNA metabolism. RPA binds with high affinity to single-stranded DNA and interacts with multiple proteins, including proteins required for the initiation of SV40 DNA replication, DNA polymerase alpha and SV40 large T antigen. We have used a series of mutant derivatives of RPA to map the regions of RPA required for specific protein-protein interactions and have examined the roles of these interactions in DNA replication. T antigen, DNA polymerase alpha and the activation domain of VP16 all have overlapping sites of interaction in the N-terminal half (residues 1-327) of the 70-kDa subunit of RPA. In addition, the interaction site for DNA polymerase alpha is composed of two functionally distinct regions, one (residues 1- approximately 170) which stimulates polymerase activity and a second (residues approximately 170-327) which increases polymerase processivity. In the latter, both the direct protein-protein interaction and ssDNA-binding activities of RPA were needed for RPA to modulate polymerase processivity. We also found that SV40 T antigen inhibited the ability of RPA to increase processivity of DNA polymerase alpha, suggesting that this activity of RPA may be important for elongation but not during the initiation of DNA replication. DNA polymerase alpha, but not T antigen also interacted with the 32- and/or 14-kDa subunits of RPA, but these interactions did not seem to effect polymerase activity.
Braun
K A
KA
Department of Biochemistry, University of Iowa College of Medicine, 51 Newton Road, Iowa City, Iowa 52242-1109, USA.
Lao
Y
Y
He
Z
Z
Ingles
C J
CJ
Wold
M S
MS
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism / Q000494:pharmacology; D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005453:Fluorescence; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D051716:Replication Protein A
D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65; C497167:RPA1 protein, human; D051716:Replication Protein A; D004257:DNA Polymerase II