RecQ helicases play key roles in maintaining genome stability and preventing early onset of aging. However, their exact mechanisms of action remain unclear. One reason is that the functional units of these helicases in vivo—the endogenous complexes containing them—have not been purified and studied. We report here three human complexes containing the BLM helicase involved in BS and demonstrate that they have distinct subunit compositions. One complex contains five FA proteins, whereas another contains MLH1, a protein involved in mismatch repair and mutated in colon cancer. Identification of these distinct complexes permits further biochemical characterization of their roles in cellular pathways and in disease etiology.
The most important finding of the present study is the observation that five FA proteins are part of a BLM complex that possesses a DNA-unwinding activity. To our knowledge, this is the first biochemical activity for a complex containing the endogenous FA proteins. Our data imply that these FA proteins may have a function in DNA repair as part of this BLM-dependent DNA-unwinding complex. Thus far, the molecular function of the FA core complex proteins remains unknown. They have been proposed to function in at least two distinct pathways: DNA repair and detoxification of reactive oxygen species (4
). More-recent evidence suggests that these FA proteins control the activation of FANCD2 by monoubiquitination, a step required for DNA-damage-dependent colocalization with BRCA1 (12
). Our Superose-6 analysis shows that FANCA is present in at least three nuclear complexes, only one of which contains BLM (Fig. ). It will be important to determine the composition of the other two FANCA-containing complexes, as well as their function.
Although BS and FA cells have strikingly different phenotypes, with elevated sister chromatid exchanges in the former and cross-linker-induced quadriradials in the latter, a connection between the two diseases may be inferred from several lines of earlier evidence. First, both conditions feature genomic instability, predisposition to cancer, and reduced fertility. Second, the type of spontaneous chromosomal instability in FA was noted to be similar to that of BS (21
), showing mainly chromatid breaks and interchanges. This is in contrast to other caretaker gene disorders that favor deletions, rearrangements, and variable chromosomal numbers. The similarity also extends to the chromosomal aberration observed in BRCA2-deficient cells, which was found to be similar to that in both BS and FA cells (37
), long before BRCA2 was identified as an FA gene itself (18
). Third, some FA cells have elevated homologous recombination activity (40
) and a higher frequency of sister chromatid exchanges (9
), which are reminiscent of BS cells. Fourth, certain BS cells exhibit increased sensitivity to MMC (17
), a hallmark of FA cells.
Our data link BS and FA by showing that BLM and five FA proteins are components of a single complex. The findings that this complex includes at least three DNA-binding and remodeling proteins (BLM helicase, Topo IIIα, and RPA) and possesses a DNA-unwinding activity suggest that it could participate in the removal of aberrant DNA structures formed during dynamic processes such as DNA replication and repair. In accord with this notion, BLM helicase is capable of unwinding several types of DNA structures (3
). Topo IIIα could facilitate unwinding by relieving the torsional stress generated by the helicase. RPA may play one or both roles in this process: it may participate in recognition of the DNA damage, just as it does for nucleotide excision repair, or it may facilitate the unwinding reaction by stabilizing single-stranded DNA generated by the BLM helicase. The single-stranded DNA thus generated could then serve as a critical intermediate in recombination or repair. Malfunction of proteins in this complex could result in the accumulation of aberrant DNA structures that lead to chromosomal aberrations observed in these diseases.
An important unanswered question is how BLM and FA proteins function together in the BRAFT complex. Currently, there is no biochemical activity described for the core complex FA proteins, which makes it difficult to establish a functional connection between BLM and FA proteins. Our ubiquitination analyses suggest that these proteins do not function in a linear signaling pathway through monoubiquitination (Fig. ). However, our ongoing work has suggested a new possibility: that core complex FA proteins, or their associated FAAPs, may possess DNA-modifying activities that are complementary to those present in BLM, Topo IIIα, and RPA during unwinding of the damaged DNA. In other words, FA proteins and BLM may not regulate each other's activity but instead may modify DNA structures in concert in sequential steps during the DNA repair process. Indeed, we found that one of the uncharacterized FAAPs contains a DNA endonuclease domain highly homologous to that of a protein known to be involved in DNA damage repair (unpublished data). Speculatively, FA proteins, through this FAAP, may provide a DNA-cutting activity necessary for the subsequent unwinding step catalyzed by the BLM helicase. Future experiments will be needed to analyze whether BRAFT has multiple DNA-modifying activities that cooperate to unwind the damaged DNA.