Cisplatin-DNA binding specificity of calf high-mobility group 1 protein.
We have identified a series of proteins with an affinity for cisplatin -damaged DNA using damaged DNA affinity chromatography. We have purified one of these proteins to homogeneity on the basis of a mobility shift assay detecting binding to cisplatin-damaged DNA. The protein was identified as high-mobility group 1 protein (HMG-1) by N-terminal protein sequence analysis. Analysis of a variety of DNA structures revealed that fully duplex DNAs were the best substrates for HMG-1 binding, while partial duplexes were less avidly bound. The decreased levels of binding are attributed to the length of the duplex region of the DNA substrates. A 3-fold increase in binding was observed when a cisplatin-damaged DNA substrate containing a single break in the phosphodiester backbone was joined by DNA ligase. The strict DNA size dependence of binding was also assessed, and a 10-fold increase in binding was observed when the length of the DNA duplex was increased from 44 to 180 base pairs (bp) at the same level of cisplatin damage. HMG-1 binding also was correlated with the degree of cisplatin-DNA damage, suggesting a higher affinity for DNA containing multiple cisplatin adducts. Nuclease degradation of the cisplatin-damaged DNA demonstrated that at the lowest levels of cisplatin damage all of the substrates contained at least one cisplatin adduct. The potential role of HMG-1 in the repair of cisplatin-DNA adducts is discussed.
Turchi
J J
JJ
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio 45435, USA.
Li
M
M
Henkels
K M
KM
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002850:Chromatography, Gel; D002945:Cisplatin / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000378:metabolism
D018736:DNA Adducts; D006609:High Mobility Group Proteins; D009838:Oligodeoxyribonucleotides; C057270:cisplatin-DNA adduct; D002945:Cisplatin