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Weisshart, K.* ; Pestryakov, P.* ; Smith, R.W.P.* ; Hartmann, H.* ; Kremmer, E. ; Lavrik, O.* ; Nasheuer, H.-P.*

Coordinated regulation of replication protein A activities by its subunits p14 and p32.

J. Biol. Chem. 279, 35368-35376 (2004)
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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, RPAΔp14, consisting of p70 and p32. RPAΔp14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPAΔp14 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 RPAΔp14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase α-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.
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Publication type Article: Journal article
Document type Scientific Article
Language english
Publication Year 2004
HGF-reported in Year 0
ISSN (print) / ISBN 0021-9258
e-ISSN 1083-351X
Quellenangaben Volume: 279, Issue: , Pages: 35368-35376 Article Number: , Supplement: ,
Publisher American Society for Biochemistry and Molecular Biology
Reviewing status Peer reviewed
PSP Element(s) G-501700-003
Erfassungsdatum 2004-10-22