Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains.
The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein-DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5'-protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a 'zero-length' photoreactive group (4-thio-2'-deoxyuridine-5'-monophosphate) embedded in the 3' end of the DNA primer, or a group connected to the 3' end by a lengthy linker (5-[N-[N-(4-azido-2,5-difluoro-3- chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C.p32D.p14) was capable of correctly recognizing the primer- template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D.p14) was incapable of detectably recognizing the DNA- junction structures, suggesting an orchestrating role for p70C in this process.
Pestryakov
Pavel E
PE
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Prospect Lavrentieva 8, 630090, Novosibirsk, Russia.
Khlimankov
Denis Y
DY
Bochkareva
Elena
E
Bochkarev
Alexey
A
Lavrik
Olga I
OI
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D013698:Templates, Genetic
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D021122:Protein Subunits; C497167:RPA1 protein, human; D051716:Replication Protein A