Coordinated regulation of replication protein A activities by its subunits p14 and p32.
The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPADeltap14, consisting of p70 and p32. RPADeltap14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADeltap14 complex p32 only minimally recognized the 3'-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPADeltap14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication.
Weisshart
Klaus
K
Institute of Molecular Biotechnology, Beutenbergstrasse 11, Jena 07745, Germany.
Pestryakov
Pavel
P
Smith
Richard W P
RW
Hartmann
Hella
H
Kremmer
Elisabeth
E
Lavrik
Olga
O
Nasheuer
Heinz-Peter
HP
D000957:Antigens, Viral, Tumor / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D012097:Repressor Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D000957:Antigens, Viral, Tumor; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C490043:IFRD2 protein, human; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D012097:Repressor Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I