High-mobility group 1 protein inhibits helicase catalyzed displacement of cisplatin-damaged DNA

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Abstract

We have determined the effect of HMG-1 bound to cisplatin-damaged DNA on the activities of calf helicase E. DNase I protection analysis demonstrated HMG-1 bound a cisplatin-damaged 24 base oligonucleotide annealed to M13mp18. Exonuclease digestion experiments revealed that greater than 90% of the DNA substrates contained a single site specific cisplatin adduct and, maximally, 65% of the substrates were bound by HMG-1. Helicase E catalyzed displacement of the cisplatin-damaged DNA oligonucleotide was inhibited by HMG-1 in a concentration-dependent manner. Time course experiments revealed a decreased rate of displacement in reactions containing HMG-1. The maximum inhibition observed was 55% and taking into account that only 65% of the substrates had HMG-1 bound, approximately 85% inhibition was observed on platinated DNA substrates containing HMG-1. Inhibition of helicase activity was proportional to the amount of substrate bound by HMG-1 based on the displacement and exonuclease assays at varying HMG-1 concentrations. The ability of helicase E to displace an undamaged DNA oligonucleotide from a cisplatin-damaged DNA template was also inhibited by HMG-1. Interestingly, HMG-1 had no effect on the rate of DNA-dependent ATP hydrolysis catalyzed by helicase E on the same DNA substrate. The inhibition of helicase activity by HMG-1 binding cisplatin-damaged DNA further supports a role for HMG-1 inhibiting DNA repair which may contribute to cellular sensitivity to cisplatin.

Introduction

The chemotherapeutic drug cis-diamminedichloroplatinum(II) (cisplatin) has been used extensively in the treatment of a variety of cancers. A major clinical limitation of cisplatin treatment for ovarian cancer is that following tumor regression and remission, recurrence and resistance often occur 1, 2. The cytotoxic action of cisplatin is generally believed to involve the formation of covalent DNA-adducts which block enzymes involved in replication and transcription [3]. This, however, cannot entirely explain the cytotoxic action of cisplatin since transplatin, the trans isomer of cisplatin, can form DNA-adducts but remains clinically ineffective 4, 5.

The metabolism of cisplatin–DNA adducts is not completely understood, but several damage recognition proteins (DRPs) have been shown to bind and recognize cisplatin-damaged DNA, including HMG-domain proteins 6, 7, 8, 9. The human nucleotide excision repair pathway can repair cisplatin–DNA adducts but it is inefficient compared to the repair of UV induced DNA damage [10]. Enhanced DNA repair of cisplatin–DNA adducts has been demonstrated to play a role in cisplatin resistance 11, 12, 13, 14. The cytotoxic action of cisplatin may be potentiated by DRPs binding cisplatin–damaged DNA sites, blocking repair, and thus sensitizing cells to cisplatin treatment [15]. This hypothesis is consistent with the inability of HMG-1 to recognize and bind transplatin-damaged DNA 9, 16. HMG-1 has been shown to recognize and bind cisplatin-damaged DNA, and the binding has been characterized with respect to DNA structure and degree of cisplatin damage 9, 17.

Calf thymus helicase E is an ATP-dependent DNA helicase that has been proposed to have a role in DNA repair based on enzymatic properties 18, 19, 20. Helicase E can interact with polymerase ϵ in a coordinated displacement synthesis reaction [20]. Helicase E has also been shown to displace an oligonucleotide damaged with cisplatin [19]. These enzymatic properties are consistent with helicase E playing a role in cisplatin–DNA repair.

To address the hypothesis that HMG-1 inhibits the repair of cisplatin–DNA adducts, we have developed an in vitro model system to assay the effect HMG-1 has on helicase catalyzed DNA displacement. Using purified proteins and a synthetic DNA substrate, we have demonstrated that HMG-1 binding to cisplatin-treated DNA resulted in a significant reduction of helicase catalyzed displacement activity. Our results demonstrate that the degree of inhibition is proportional to the portion of DNA substrates with HMG-1 bound. These results are discussed with respect to the repair of cisplatin–DNA adducts and the cytotoxic action of cisplatin.

Section snippets

Materials

Unlabeled nucleotides were purchased from Boehringer Mannheim (Indianapolis, IN) and radiolabeled nucleotides were from Dupont NEN (Boston, MA). T7 DNA polymerase (form II unmodified) and T4 polynucleotide kinase were from New England Biolabs (Beverly, MA). Sequenase (ver. 2.0), E. coli SSB, and DNase I were from United States Biochemical (Cleveland, OH). Cisplatin was from Sigma (St. Louis, MO). All other reagents were purchased from standard suppliers.

Enzymes

Helicase E was purified from calf thymus

Results

A DNA substrate was designed to measure the effect of bound HMG-1 on helicase catalyzed displacement of a cisplatin-damaged oligonucleotide. The substrate consisted of a DNA oligonucleotide 24 bases in length containing a site specific 1,2d(GpG) cisplatin adduct annealed to ssM13mp18. We first assessed HMG-1 binding to the cisplatin-damaged and control DNA substrates in DNase I protection analyses. The results demonstrate that HMG-1 binds to the cisplatin-damaged substrate (Fig. 1A), while no

Discussion

In this report, we have determined how the metabolism of cisplatin–DNA adducts is altered by HMG-1. HMG-1 has been demonstrated to bind cisplatin-damaged DNA in vitro using both a damaged-DNA cellulose precipitation assay and in mobility shift assays 8, 9, 17. Recent results demonstrated a change in the intracellular localization of HMG-1 in cells treated with cisplatin, suggesting that HMG-1 may bind DNA damaged by cisplatin in vivo [27]. Ixr1 encodes a yeast HMG box protein, and null mutants

Acknowledgements

This work was supported by a National Institutes of Health award CA64374 to J.J.T. S.M.P. is supported by a predoctoral fellowship from the Wright State University Biomedical Sciences PhD program.

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