Interaction of replication protein A with photoreactive DNA structures.
A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, Novosibirsk 630090, Russia.
N A
NA
Lebedeva
E A
EA
Mal'tseva
I Yu
IY
Garipova
S V
SV
Vasil'eva
E A
EA
Belousova
I O
IO
Petruseva
N I
NI
Rechkunova
V N
VN
Sil'nikov
O I
OI
Lavrik
D001386:Azides / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001565:Benzoates / Q000737:chemistry*; D001665:Binding Sites; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry*; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051716:Replication Protein A; D013379:Substrate Specificity
C110129:4-azidotetrafluorobenzoic acid; D001386:Azides; D001426:Bacterial Proteins; D001565:Benzoates; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009711:Nucleotides; D009841:Oligonucleotides; D051716:Replication Protein A