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.Germ line mutations in BRCA1 lead to an increased lifetime risk of breast and/or ovarian cancer. Cellular studies have revealed that the BRCA1 tumor suppressor gene is required for the maintenance of genomic integrity and a normal level of resistance to DNA damage (for review see Refs. 1-3). The nuclear phosphoprotein BRCA1 contains tandem C-terminal BRCT motifs, a conserved protein sequence found in a large number of DNA damage-response proteins (4). The integrity of the BRCT motifs is required for the role of BRCA1 in double strand break repair (DSBR) 1 and homologous recombination (5-8). Tumor-predisposing missense and deletion mutations in the BRCA1 BRCT domain, all of which render BRCA1 defective in its DSBR function, also disrupt the ability of BRCA to bind BACH1. BACH1 is a member of the DEAH subfamily of superfamily 2 helicases (9). Consistent with its predicted helicase domain, BACH1 was recently shown to catalyze DNA unwinding of M13 partial duplex substrates and have a 5Ј to 3Ј polarity on a linearized M13 directionality substrate (10). 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). More recently, it was shown that the interaction between BRCA1 and BACH1 depends on the phosphorylation status of BACH1 (11-13), and that this phosphorylationdependent interaction is required for DNA damage-induced checkpoint control during the G 2 /M phase of the ...

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Mutations in the FANCJ helicase predispose individuals to breast cancer and are genetically linked to the Fanconi anemia (FA) complementation group J. FA is a chromosomal instability disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination pathway of double strand break repair. In an effort to understand the molecular functions of FANCJ, we have investigated the ability of purified FANCJ recombinant protein to use its motor ATPase function for activities in addition to unwinding of conventional duplex DNA substrates. These efforts have led to the discovery that FANCJ ATP hydrolysis can be used to destabilize protein-DNA complexes and unwind triple helix alternate DNA structures. These novel catalytic functions of FANCJ may be important for its role in cellular DNA repair, recombination, or resolving DNA structural obstacles to replication. Consistent with this, we show that FANCJ can inhibit RAD51 strand exchange, an activity that is likely to be important for its role in controlling DNA repair through homologous recombination.A growing interest in the mechanisms of helicases in cellular nucleic acid metabolism has been partly fueled by the knowledge that an increasing number of human diseases are genetically linked to mutations in genes encoding helicase-like proteins. RecQ helicases are prominent in this category, since three diseases of premature aging and/or cancer are attributed to recessive mutations in genes that encode bona fide DNA helicases (Werner and Bloom Syndromes) or DNA-dependent ATPase (Rothmund-Thomson Syndrome) (1, 2). Other helicase disorders exist, including Fanconi anemia (FA), 3 which is a recessive genetic disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high risk of cancer (for a review, see Ref.3). Among the 13 FA complementation groups from which all of the FA genes have been cloned, the FANCM and FANCJ genes encode DNA-stimulated ATPases, the latter also being a Superfamily 2 DNA helicase (4). The interaction of FANCJ with BRCA1 (5) and the existence of FANCJ mutations in early onset breast cancer patients (5, 6) as well as its genetic linkage to the FA-J complementation group (7-9) have clarified that FANCJ is a tumor suppressor.Although FANCJ helicase activity has been studied biochemically, the cellular functions of FANCJ in DNA repair are likely to require additional functions that involve protein-protein and protein-DNA interactions. Two protein partners of FANCJ that appear to be important are the single-stranded DNA-binding protein RPA, which stimulates FANCJ helicase activity (10), and the mismatch repair complex MutL␣, to which FANCJ binds and enables FANCJ to perform its DNA repair function (11). The FANCJ helicase functions downstream of FANCD2 monoubiquitination, a critical event in the FA pathway (9...