Recognition of chemical carcinogen-modified DNA by a DNA-binding protein.

Abstract

Using a filter binding assay, we have detected and partially purified a protein from human placenta that has a high affinity for N-acetoxy-2-acetylaminofluorene-modified double-stranded DNA (AAF-[3HJDNA) of bacteriophage 17. This protein has been partially purified from a 1 M NaC extract of a crude nuclear fraction by a combination of ion-exchange and nucleic acid affinity chromatography. With Although the excision repair of chemical damage clearly shares many features with the repair of UV-induced damage (1), recent experiments have suggested that there may be differences in the ways in which lesions produced by these two types of agents are recognized and removed (2, 3). If differences do exist, a class of proteins may exist that selectively recognizes DNA modified by chemical carcinogens. This logic has led us to search for DNA-binding proteins that recognize N-acetoxy-2-acetylaminofluorene (NA-AAF)modified DNA (AAF-DNA).To assay for such proteins, we have used filter binding techniques (4) similar to those used to demonstrate DNA-binding proteins that specifically recognize UV-damaged DNA (5) and partially depurinated DNA (6). In this paper we report the partial purification and characterization of a DNA-binding protein from human placenta that shows strong preferential binding to AAF-DNA and DNA modified with methyl methanesulfonate or methylnitrosourea (MMS-DNA or MNU-DNA, respectively) as compared to single-and double-stranded DNA, supercoiled bacteriophage OX174 DNA, partially depurinated DNA, glucosylated bacteriophage T4 DNA, or UV-irradiated DNA.MATERIALS AND METHODS Preparation of Binding Substrates. Bacteriophage T7[3H]DNA (1.21 X 105 cpm/,ug, unless otherwise indicated) was prepared from phage grown and isolated as described in the literature (7,8), and the DNA was purified from NaDoDSO4-lysed, proteinase K-digested (Merck) phage (9).AAF-[3H]DNA was prepared as described (10).

A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells.

“…According to its DNA-binding characteristics and substrate specificity, the DDB protein complex we identified appears to be different from the DDB proteins previously isolated from HeLa cells (16,34) or human placenta (10,25) and from other types of DNA-binding proteins such as histones and transcription factors. This protein complex also appears to belong to a different class from those described by Glazer et al (14), who found UV-inducible DNA-binding proteins in HeLa cells in the presence of either dactinomycin or cycloheximide and which have no specificity for UVdamaged DNA.…”

“…Complementation analysis of cells from patients with DNA repair disorders, such as xeroderma pigmentosum (XP), has indicated that the repair of bulky DNA lesions in mammalian cells is considerably more complex than that in bacteria. Several damage-specific DNA-binding (DDB) proteins in mammalian cells and tissues have been described (8-10, 25,34), but the physiological roles of these proteins have not been determined. Since isolation and cloning of the genes which encode mammalian DNA repair proteins have met with little success, specific information about the formation of DNA repair complexes (in particular those involved in the repair of bulky lesions, such as UV photoproducts) and their regulation in a stress-induced environment is not yet available.…”

An ultraviolet light-damaged DNA recognition protein absent in xeroderma pigmentosum group E cells binds selectively to pyrimidine (6–4) pyrimidone photoproducts

“…Several studies have revealed the existence of proteins in mammalian cells that bind specifically to DNA that has been damaged by chemical or physical agents. DNA damage recognition proteins (DRPs) have been reported for a growing list of DNA modifications, including certain cisplatin crosslinks (1,2), 1 ,N6-ethenoadenine (3), G:T mismatches (4), apurinic/apyrimidinic (AP) sites (5), N-acetyl-2-aminofluoreneguanine adducts (6), and ultraviolet light (UV)-induced photoproducts (7-9). The cellular function of these DRPs is currently unknown, but it is reasonable to speculate that some or all of these proteins may play roles in DNA repair (8,9), or in other biological activities related to the genotoxicity of the agent under investigation (1,2).…”

A damage-recognition protein which binds to DNA containing interstrand cross-links is absent or defective in Fanconi anemia, Complementation group A, cells

“…Damage recognition is an important component of the initial steps in the DNA repair process. A number of damage recognition proteins have been reported which bind to types of damaged DNA other than those containing psoralen plus UVA light induced interstrand cross-links (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). None of these proteins has been found to have DNA endonucleolytic activity but many have been hypothesized to play a role in the repair process.…”

“…Proteins which specifically recognize and bind to DNA containing interstrand cross-links and which have been shown to play a role in the repair process have not been identified in mammalian cells. DNA binding proteins which recognize other types of DNA damage such as base modifications produced by either chemical or physical agents (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), bulky lesions (16)(17)(18), abasic sites (19,20), and mismatches in DNA (21), have been described in mammalian cells but whether these proteins play a role in the DNA repair processes is not clear, though has been suggested by the results of several of the studies.…”