Preferential binding of high mobility group 1 protein to UV-damaged DNA. Role of the COOH-terminal domain.
Binding of chromosomal high mobility group 1 protein (HMG1) to UV-damaged DNA has been studied with oligonucleotides containing a single dipyrimidine site for formation of UV photolesions. Irradiation of an oligonucleotide with unique TT dinucleotide resulted in generation of cyclobutane pyrimidine dimer with no evidence for induction of (6-4) photoproducts, whereas the analysis of irradiated TC-containing oligonucleotide detected (6-4) photoproducts but not cyclobutane pyrimidine dimers. Mobility shift assays have revealed that HMG1 protein binds preferentially to irradiated TT and TC oligonucleotides. Photoreversal of cyclobutane pyrimidine dimers with DNA photolyase and hydrolysis of the (6-4) photoproducts with hot alkali substantially reduced but did not eliminate binding of HMG1. The protein, therefore, appears to bind the two main types of UV damages in DNA, but some other photolesion(s) contributes to the preferential binding of HMG1 to irradiated DNA. By quantifying gel shift assays and considering the efficiencies of lesion formation, we determined dissociation constants of 1.2 +/- 0.5 and 4.0 +/- 1.5 microM for irradiated TT and TC oligonucleotides, respectively, and 70 +/- 20 microM for the control non-irradiated probes. Tryptic removal of the acidic COOH-terminal domain of HMG1 significantly affected binding of the protein to both irradiated and intact oligonucleotides. The potential role of HMG1 in recognition of the UV lesions in DNA is discussed.
Pasheva
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EA
Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. eva@obzor.bio21.bas.bg
Pashev
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IG
Favre
A
A
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000378:metabolism / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013950:Thymus Gland / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D004273:DNA, Neoplasm; D006609:High Mobility Group Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA