ABSTRACT
Proteins recognizing and binding to damaged DNA (DDB-proteins) were analyzed in human lymphocytes obtained from healthy donors. Using an electrophoretic mobility shift assay several complexes between nuclear extract proteins and damaged DNA were detected: a complex specific for DNA damaged by N-acetoxy-N-acetylaminofluorene, another complex specific for UV-irradiated DNA, and two complexes specific for DNA damaged by cis-dichlorodiammine platinum. All the detected complexes differed in electrophoretic mobility and possibly contained different proteins. Complexes specific for free DNA ends were also detected in protein extracts from lymphocytes.
Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , Lymphocytes/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity , Acetoxyacetylaminofluorene/toxicity , Adult , Base Sequence , Cell Nucleus/metabolism , Cells, Cultured , Cisplatin/toxicity , Cytoplasm/metabolism , DNA-Binding Proteins/isolation & purification , Humans , Lymphocytes/drug effects , Lymphocytes/radiation effects , Oligonucleotide Probes , Ultraviolet RaysABSTRACT
High mobility group (HMG) proteins 1 and 2 are abundant non-histone chromosomal proteins that bind preferentially DNA that is bent or underwound. Previous studies have shown that these proteins preferentially bind to DNA damaged by the crosslinking agents cis-diammine-dichloro-platinum(II), chromium(III) and UV-C radiation. Here we have studied the binding of HMG-1/2 proteins to a duplex oligonucleotide damaged by benzo(a)pyrene diol epoxide or N-acetoxy-acetylaminofluorene using an electrophoretic mobility shift assay. Both chemicals induce monoadducts that are known to distort DNA structure. The affinities of HMG-1/2 for DNA damaged by benzo[a]pyrene diol epoxide or N-acetoxy-acetylaminofluorene were similar to that for UV-irradiated DNA, which were an order of magnitude higher than for undamaged DNA. In contrast, DNA modified by dimethyl sulfate was not preferentially recognised by HMG-1/2.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/pharmacology , Acetoxyacetylaminofluorene/pharmacology , DNA Adducts/metabolism , High Mobility Group Proteins/metabolism , Alkylating Agents/pharmacology , Animals , Binding Sites , Blotting, Southern , Carcinogens/pharmacology , DNA Probes/chemistry , DNA Probes/drug effects , DNA Probes/metabolism , DNA Probes/radiation effects , Male , RatsABSTRACT
Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.
