Polarity of human replication protein A binding to DNA.
Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3'-end of the upstream oligonucleotide or at the 5'-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5'-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3'-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer-template systems containing 5'- or 3'-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.
Kolpashchikov
D M
DM
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, Prospect Lavrentiev 8, 630090, Novosibirsk, Russia.
Khodyreva
S N
SN
Khlimankov
D Y
DY
Wold
M S
MS
Favre
A
A
Lavrik
O I
OI
D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication / Q000528:radiation effects; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000528:radiation effects; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000528:radiation effects; D051716:Replication Protein A; D014466:Ultraviolet Rays; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry
C110508:5-(N-(2-nitro-5-azidobenzoyl)-3-aminopropen-1-yl)deoxyuridine-5'-triphosphate; D001386:Azides; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; C497167:RPA1 protein, human; D051716:Replication Protein A; D014544:Uridine Triphosphate