Analysis of the DNA Substrate Specificity of the Human BACH1 HelicaseAssociated with BreastCancer

Rigu Gupta,Sudha Sharma,Joshua A Sommers,Zhe Jin,Sharon B Cantor,Robert M Brosh

doi:10.1074/jbc.m501995200

Abstract

We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.

Highlights

From the ‡Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224 and the §Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
DNA unwinding studies indicate that certain DNA helicases preferentially unwind forked duplex substrates [15, 18, 19]; we examined BACH1 helicase activity on a related forked duplex substrate that was flanked by 5Ј and 3Ј single-stranded DNA (ssDNA) tails of 26 and 25 nt, respectively (Table II, substrate 15)
These results indicate that BACH1 preferentially unwinds a forked duplex substrate with 3Ј and 5Ј ssDNA arms compared with a substrate flanked by only a 5Ј ssDNA tail

Summary

Introduction

From the ‡Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224 and the §Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605. BACH1 requires nucleic acid continuity in the 5؅ ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination. A role of BACH1 helicase in DSBR was suggested by the observation that overexpression of a BACH1 allele (K52R) carrying a mutation in its ATP-binding pocket that inactivates its ATPase/ helicase function [10] resulted in a marked decrease in the ability of cells to repair DSBs, and that this dominant negative phenotype depended on a specific interaction between BACH1 and BRCA1 [9]. The evidence linking BACH1 with the hereditary breast cancer gene BRCA1 and the identification of patients with mutations in the BACH1 gene itself are consistent with the hypothesis that BACH1 helicase functions in tumor suppression

Methods

Results

Conclusion