Mechanistic insight into cadmium-induced inactivation of the Bloom protein.

Wei Qin,Etienne Henry,Eric Deprez,Xu-Guang Xi,Nicolas Bazeille,Bo Zhang

doi:10.1038/srep26225

Abstract

Cadmium is a toxic metal that inactivates DNA-repair proteins via multiple mechanisms, including zinc substitution. In this study, we investigated the effect of Cd2+ on the Bloom protein (BLM), a DNA-repair helicase carrying a zinc-binding domain (ZBD) and playing a critical role to ensure genomic stability. One characteristics of BLM-deficient cells is the elevated rate of sister chromatid exchanges, a phenomenon that is also induced by Cd2+. Here, we show that Cd2+ strongly inhibits both ATPase and helicase activities of BLM. Cd2+ primarily prevents BLM-DNA interaction via its binding to sulfhydryl groups of solvent-exposed cysteine residues and, concomitantly, promotes the formation of large BLM multimers/aggregates. In contrast to previously described Cd2+ effects on other zinc-containing DNA-repair proteins, the ZBD appears to play a minor role in the Cd2+-mediated inhibition. While the Cd2+-dependent formation of inactive multimers and the defect of DNA-binding were fully reversible upon addition of EDTA, the inhibition of the DNA unwinding activity was not counteracted by EDTA, indicating another mechanism of inhibition by Cd2+ relative to the targeting of a catalytic residue. Altogether, our results provide new clues for understanding the mechanism behind the ZBD-independent inactivation of BLM by Cd2+ leading to accumulation of DNA double-strand breaks.

Highlights

Bloom’s syndrome (BS) is a rare, autosomal and recessive disease resulting from the mutational inactivation of a human RecQ family helicase encoded by the blm gene[1]
These results, suggesting that Bloom protein (BLM) represents a more sensitive target for Cd2+, were confirmed by stopped-flow FRET experiments allowing measurements of the unwinding kinetic rate constant of BLM by using partial duplex DNA labeled with fluorescein and hexachlorofluorescein as a donor and acceptor, respectively[29,30] (Supplementary Fig. S1; see Supplementary Table S1 for details about the structure of the DNA substrate): the levels of Cd2+-dependent inhibition of BLMfull-length and BLM642−1290 helicase activities were similar and significantly higher than that observed with RecQE.coli (≈10-fold higher)
Recent studies highlight that Cd2+ targets major players of the DNA-repair machinery including proteins involved in the BER, NER or Mismatch Repair (MMR) pathways[21,22,23], little is known about Cd2+ effects on proteins involved in the double-strand break repair (DSB) pathway such as BLM

Summary

Introduction

Bloom’s syndrome (BS) is a rare, autosomal and recessive disease resulting from the mutational inactivation of a human RecQ family helicase encoded by the blm gene[1]. Many in vitro studies on Cd2+-mediated toxic effects have been performed with proteins involved in the Base and Nucleotide Excision Repair (BER/NER)[20,21], Mismatch Repair (MMR)[22,23] and Non-Homologous End-Joining (NHEJ)[24], it is a difficult task to highlight a general/common mechanism underlying Cd2+-mediated inhibition of DNA-repair systems It appears that detrimental effects of Cd2+ on DNA-repair proteins occur through the binding of Cd2+ to functional sulfhydryl groups[23,25], and the replacement of Zn2+ by Cd2+ in ZBDs represents one cause for protein dysfunctions. Based on previous observations showing inhibitory effects of Cd2+ on zinc-containing DNA-repair proteins and taking into account characteristic phenotypes of Cd2+-exposed human and yeast cells[12], we addressed in the present study the molecular mechanisms of Cd2+-dependent BLM inactivation. Surface and solvent-exposed Cys mediate the Cd2+-dependent formation of aggregates (reversible by EDTA) whereas at least one additional residue, playing a key role in the catalytic process, is targeted by Cd2+ in an irreversible manner

Methods

Results

Conclusion