Inhibition of BACH1 (FANCJ) helicase by backbone discontinuity is overcome by increased motor ATPase or length of loading strand

The BRCA1 associated C-terminal helicase (BACH1) associated with breast cancer has been implicated in double strand break (DSB) repair. More recently, BACH1 (FANCJ) has been genetically linked to the chromosomal instability disorder Fanconi Anemia (FA). Understanding the roles of BACH1 in cellular DNA metabolism and how BACH1 dysfunction leads to tumorigenesis requires a comprehensive investigation of its catalytic mechanism and molecular functions in DNA repair. In this study, we have determined that BACH1 helicase contacts with both the translocating and the non-translocating strands of the duplex are critical for its ability to track along the sugar phosphate backbone and unwind dsDNA. An increased motor ATPase of a BACH1 helicase domain variant (M299I) enabled the helicase to unwind the backbone-modified DNA substrate in a more proficient manner. Alternatively, increasing the length of the 5′ tail of the DNA substrate allowed BACH1 to overcome the backbone discontinuity, suggesting that BACH1 loading mechanism is critical for its ability to unwind damaged DNA molecules

BRCA-FA pathway as a target for anti-tumor drugs

Promising research on DNA repair signaling pathways predicts a new age of anti-tumor drugs. This research was initiated through the discovery and characterization of proteins that functioned together in signaling pathways to sense, respond, and repair DNA damage. It was realized that tumor cells often lacked distinct DNA repair pathways, but simultaneously relied heavily on compensating pathways. More recently, researchers have begun to manipulate these compensating pathways to reign in and kill tumor cells. In a striking example it was shown that tumors derived from mutations in the DNA repair genes, of BRCA-FA pathway, were selectively sensitive to inhibition of the base excision repair pathway. These findings suggest that tumors derived from defects in DNA repair genes will be easier to treat clinically, providing a streamlined and targeted therapy that spares healthy cells. In the future, identifying patients with susceptible tumors and discovering additional DNA repair targets amenable to anti-tumor drugs will have a major impact on the course of cancer treatment

Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer

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 \u27 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 \u27 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

FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein

The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and hereditary breast cancer. A critical role of FANCJ helicase may be to restart replication as a component of downstream events that occur during the repair of DNA cross-links or double-strand breaks. We investigated the potential interaction of FANCJ with replication protein A (RPA), a single-stranded DNA-binding protein implicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a direct interaction of FANCJ and the RPA70 subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with RPA in nuclear foci after DNA damage. Our data are consistent with a model in which FANCJ associates with RPA in a DNA damage-inducible manner and through the protein interaction RPA stimulates FANCJ helicase to better unwind duplex DNA substrates. These findings identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of the helicase to more efficiently unwind DNA repair intermediates to maintain genomic stability

FANCJ Uses Its Motor ATPase to Destabilize Protein-DNA Complexes, Unwind Triplexes, and Inhibit RAD51 Strand Exchange*

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