The molecular and genetic functions of the BACH1 helicase in DNA repair and the molecular pathology of FA due to helicase dysfunction remain to be fully characterized. In an effort to better understand the biochemical properties of BACH1, we have investigated the catalytic activities of BACH1 and two associated polymorphic variants. We have determined that BACH1 helicase interactions with the sugar phosphate backbone of both strands of the duplex are important as BACH1 tracks along the DNA molecule and separates the strands. The backbone tracking mechanism of BACH1 is distinct from that of NPH-II and other SF2 helicases suggesting that BACH1 has a specialized unwinding mechanism. Very recently, the hepatitis C virus NS3 RNA helicase (SF2) was found to readily unwind RNA duplexes that contained long stretches of polyglycol linkages in either the translocating or non-translocating strands (23
). The authors suggested that the large kinetic step size (18 bp) (24
) may enable the RNA helicase to ‘step over’ the large polyglycol lesions in its track. Measurements of the physical and kinetic step size of BACH1 helicase will be useful to understanding how this DNA helicase is completely blocked from unwinding the duplex DNA substrates with a single 18 atom polyglycol linker in either the translocating or non-translocating strands.
Previously, we reported that BACH1 helicase activity is inhibited by a polyglycol modification positioned adjacent to the duplex region in the 5′ ssDNA tail, but not the 3′ ssDNA tail, of a forked duplex substrate (13
). This result is in contrast to our findings in this study showing that BACH1 was sensitive to disruption of backbone continuity in either strand of the DNA duplex region. Thus, the effects of backbone modifications on BACH1 helicase activity may be dependent on whether the enzyme is in the initiation or elongation phases of the unwinding reaction.
Interruption of BACH1 helicase activity in the vicinity of the backbone modification on either the translocating or non-translocating strand leaves the remaining duplex (~12 bp) intact. Although there have been reports of spontaneous melting of the final 9–11 bp of duplex DNA substrates partially unwound by other helicases (25
), this was not observed in the BACH1 helicase reactions with the backbone modified DNA substrates as evidenced by the complete lack of strand displacement measured by the radiometric helicase assay. Spontaneous fraying of the partially unwound DNA substrate acted upon by BACH1 might be expected based on the theoretical thermal stability (Tm
) of the 12 bp duplex calculated from G/C content and salt concentration which is 2°C below the reaction temperature of 30°C. It is conceivable that the sequestration of BACH1 by the polyglycol obstacle or its preferential ability to bind forked DNA structures (13
) may contribute to stabilization of the remaining duplex. Unlike a number of human RecQ helicases which have been shown to catalyze strand annealing of complementary ssDNA molecules (28
), strand annealing by BACH1 is very weak (data not shown), suggesting that this activity does not contribute to the lack of complete unwinding of the polyglycol substrates by BACH1.
BACH1 partially unwound the backbone modified DNA substrates and became sequestered, indicating that BACH1 was not able to effectively unwind past the obstacle. However, by increasing the length of the 5′ ssDNA tail used for helicase loading, BACH1 was able to efficiently unwind the backbone-modified DNA substrates. This finding should be considered in light of recent studies which also indicate the importance of DNA substrate loading elements for helicase function. Increasing the length of the ssDNA overhang was shown to enhance Dda catalyzed duplex DNA unwinding (33
). Similarly, multiple NS3 helicase molecules bound to the ssDNA loading region of a partial duplex substrate are required for optimal unwinding (34
). In addition to DNA unwinding, multiple helicase molecules loaded on the same ssDNA molecule may cooperate to facilitate protein displacement (35
), an anticipated function of certain helicases. It was proposed that the presence of multiple motors may serve to prevent backward displacement on the ssDNA, resulting in an elevated force production to displace protein from the DNA molecule (35
). For BACH1, the length of the 5′ ssDNA loading tail used by the helicase during initiation has dramatic consequences for its ability to unwind past the backbone obstacle within the duplex. This may be a consequence of the loading of numerous functionally active BACH1 helicases on the DNA substrate with a longer 5′ ssDNA tail under multiple turnover conditions. Attempts to perform single turnover BACH1 helicase experiments were inconclusive since very little unwinding of the radiolabeled substrates pre-incubated with BACH1 was detected when an excess of cold competitor DNA molecule was added with ATP to initiate the reaction (data not shown). The observation that BACH1 partially unwinds the backbone modified forked duplex with a shorter 19 nt 5′ ssDNA tail and is sequestered by the backbone perturbation raises the possibility that wild-type BACH1 enzyme may be pushed forward by other BACH1 helicase molecules loaded behind it to complete unwinding.
The two BACH1 polymorphisms (P47A, M299I) exerted dramatically different effects on the catalytic activities of the respective proteins. Although the P47A mutant was found to be completely devoid of helicase activity on an M13 partial duplex substrate (9
), we detected a low residual unwinding of the preferred forked duplex substrate and a small but detectable DNA-dependent ATPase activity. BACH1-M299I helicase, on the other hand, displayed a vastly greater DNA-dependent ATPase activity compared to BACH1-WT; however, its ability to unwind unadducted forked duplexes was only improved by <2-fold under multiple turn-over conditions. BACH1-M299I also bound the forked duplex better than wild-type BACH1, suggesting that the amino acid replacement may stabilize the enzyme: DNA interaction to some extent.
Interestingly, the position of the M299I polymorphism is between the conserved helicase motifs Ia and II, a region found to be involved in ssDNA binding for the SF1 Rep helicase (36
). When the ssDNA of the Rep/ssDNA co-crystal structure is added to the super-imposed model of the SF2 HCV helicase, a potential DNA contact with a region between motifs Ia and II of HCV helicase is also observed (37
). In this region, the bacterial nucleotide excision repair helicase UvrB contains a β hairpin domain that inserts between the two DNA strands and is responsible for physically separating the strands from one another (38
). Furthermore, this motif was identified as an Iron-Sulfur metal binding domain in the XPD helicase family to which BACH1 belongs (39
) (). The Met-299 position of BACH1 resides immediately adjacent to the second of four conserved cysteine residues that compromise the metal binding domain between the Walker A (motif I) and Walker B (motif II) motifs (). It remains to be determined if BACH1 protein binds iron through its conserved metal binding domain as reported for the Sulfolobus acidocaldarius
XPD protein (39
). The notion that mutations in the Iron–Sulfur (Fe–S) domain of BACH1 are likely to be biologically important is supported by the observation that the mutation A349P in BACH1, immediately adjacent to the last cysteine of the Fe–S cluster, was identified in a patient with severe clinical symptoms of FA (11
). Mutation of Met-299 to isoleucine may perturb the local structure of BACH1 protein to dramatically alter the enzyme's ability to hydrolyze ATP.
Figure 7 BACH1 protein domains and missense mutations. (A) Cartoon depicting the BACH1 protein with the conserved helicase motifs indicated by red boxes and the positions of the metal binding domain (yellow), nuclear localization sequence (NLS), and BRCA1 interaction (more ...)
Although the helicase efficiency (i.e. number of base pairs separated per ATP hydrolyzed) is not known for BACH1, it seems likely that the increased motor ATPase function of the M299I variant improves its ability to unwind the backbone-modified DNA substrates. Moreover, the BACH1-M299I helicase is able to unwind longer DNA duplexes without modifications better than BACH1-WT (R. Gupta, S. Sharma, K. M. Doherty, J. A. Sommers, S. B. Cantor and R. M. Brosh, Jr, unpublished data), suggesting a potential significance for the improved helicase activity of the M299I variant.
The M299I allele was originally identified as a germline BACH1 coding sequence change in a patient with early-onset breast cancer (1
). Demonstration that the BACH1-M299I variant helicase has a gain of function for unwinding chemically damaged DNA substrates raises the question of how this mutation might impact the function of BACH1 in vivo
and contribute to a cancer phenotype. Our findings set a new standard that a mutation in a helicase gene of a patient may enhance the enzyme's ability to unwind damaged DNA. It is conceivable that elevated helicase activity may lead to a clinical syndrome different from that of loss of helicase activity. In the future, it will be important to characterize FA mutations to determine which class they belong. A model for BRCA1 checkpoint function at sites of DNA damage involves the assembly of a BRCA1 super-complex containing BARD1, BACH1, BRCA2 and Rad51 that is critical for the S phase checkpoint by inhibiting DNA synthesis at late-firing sites of replication initiation (40
). Dysregulated helicase activity of BACH1-M299I may be detrimental to proper maintenance of the S phase checkpoint.
A second mechanism whereby the elevated helicase activity of BACH1-M299I would be disadvantageous for genomic stability is based on the hypothesis that BACH1 serves to remove Rad51 from DNA (41
), as demonstrated for SRS2 helicase (42
). Although depletion of either BRCA1 or BACH1 disrupts HR to a similar degree, BRCA1 deficiency negates Rad51 foci formation after DNA damage whereas Rad51 foci form in BACH1-deficient cells. A model was proposed that BACH1 functions downstream of Rad51 to release Rad51 from DNA strands to complete HR (41
). BRCA1, on the other hand may down-regulate BACH1 helicase activity to prevent the untimely displacement of Rad51 from the ssDNA filaments until the strand exchange step of HR is complete. BRCA1 deficiency would result in uncontrolled and premature Rad51 dissociation by BACH1 helicase prior to the fulfillment of strand exchange. A testable model is that BACH1-M299I helicase activity fails to be regulated in an appropriate manner by BRCA1 and disrupts HR by either displacing Rad51 or the invading third stand of a D-loop intermediate at the inappropriate time. Mechanistic studies of the role of BACH1 with its protein partners in chromosomal stability will lead to further insights toward understanding how their deficiencies lead to tumourigenesis.