Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II)

Abstract

DNA modified by the antitumor drug cisdiamminedichloroplatinum(II) (cis-DDP or cisplatin) was used to identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. This factor selectively recognizes double-stranded DNA fragments modified by cis-DDP or [Pt(en)CI2J (en, ethylenediamine).Little or no binding occurs to unmodified double-stranded DNA or to DNA modified with the clinically ineffective compounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine). Low levels of binding to single-stranded DNA modified by cis-DDP are observed. The apparent molecular mass of the factor in a variety of mammalian cells is -100 kDa, as determined by modified Western blotting. Two recombinant phage have been isolated from a human B-cell Agtll library by using a cis-DDP-modified DNA restriction fragment as a probe. The two clones have insert sizes of 1.88 and 1.44 kilobases and are aligned at their 5' ends. The polypeptides encoded by the recombinant phage exhibit DNA binding properties similar to those of the cellular factor identified in crude extracts prepared from mammalian cells. Northern analysis with one ofthe clones revealed an mRNA of 2.8 kilobases that is conserved in humans and rodents. The methods used here should be applicable in studies of other damage-specific DNA binding proteins.cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a clinically important antitumor drug used for the treatment of several human cancers, especially those of genitourinary origin (1). The biological target for cis-DDP is generally accepted to be DNA (2), and considerable information is now available on binding of the drug to DNA and the structures of its major adducts (3). Despite this progress, much remains to be learned about the specificity of cis-DDP for certain tumors as well as its activity relative to other platinum compounds.Both cis-DDP and its biologically inactive trans isomer bind to DNA in vitro and in vivo, and the resultant adducts can serve as termination sites for DNA polymerase (4). DNA replication is inhibited to similar extents by both cis and trans-DDP in vivo at the same bound drug/nucleotide ratio process or repair the DNA adducts of these various platinum complexes.To address this issue, we set out to identify cellular factors that could play a role in processing DNA modified by platinum(II) complexes. Previously identified prokaryotic repair systems such as the UvrABC excision nuclease are known to excise adducts from DNA modified by cis-but not trans-DDP (6). In addition, one component of the repair complex, UvrA, possesses specific binding properties for DNA modified by damaging agents such as ultraviolet radiation (7,8). It therefore seemed reasonable that, in mammalian cells, a similar damaged DNA binding factor would exist having sufficient generality to recognize cis-DDP modified DNA as an initial step leading to repair.Our approach has involved the use of DNA modified by cis-DDP as a probe for DNA binding factors present in crude mammalian...

Identification of GAPDH as a protein target of the saframycin antiproliferative agents

“…HMG1 binding was not observed to occur with affinity reagents prepared from SafA, QAD, Pt650, nor MMC. HMG1 had previously been determined to be a potential protein target of cisplatin-DNA adducts by sequence analysis of cDNA isolated in an expression-library screening (23). Cisplatin-DNA adducts (but not transplatin-DNA adducts) were subsequently shown to bind HMG1 specifically (24); this binding has been proposed to form the chemical basis of cisplatin antitumor activity and, similarly, the lack of HMG1 binding to account for the poor clinical efficacy of transplatin in cancer therapy.…”

Cisplatin-DNA damage recognition proteins in human tumour extracts

“…Modified western blots This method of assaying for damage recognition proteins was a modification of that described (Toney et al, 1989). typically, 100 lg of extract protein was separated by SDS/PAGE (Laemmli UK, 1970) on a 5-15% gradient gel.…”

“…CDDP-DNA adducts can also be repaired in vitro by cell free extracts from mammalian cells (Hansons & Wood, 1989). Although the nucleotide excision repair system in eukaryotic cells is incompletely characterised, mammalian proteins have been identified which recognise and bind to DNA damaged by various agents including CDDP (Chu & Chang, 1988;Donahue et al, 1990;Toney et al, 1989;Chao et al, 1991c). It has been postulated that these damage recognition proteins may be involved in the initial steps of excision repair, or alternatively may block access of repair enzymes to the sites of DNA damage.…”

Interaction of FACT, SSRP1, and the High Mobility Group (HMG) Domain of SSRP1 with DNA Damaged by the Anticancer Drug Cisplatin

“…The complex is conserved across a diverse range of organisms, analogous complexes of SPT16/SSRP1 homologs having been isolated from Xenopus and Saccharomyces cerevisiae (10 -12). Although distinct roles for the two protein components of FACT have yet to be elucidated, the DNA-binding high-mobility group (HMG) domain of SSRP1 (13) may target the complex to nucleosomes (7). Circumstantial evidence suggests that HMG domain proteins bind to DNA as it enters and exits the nucleosome (14).…”