Bloom syndrome (BS) is a rare human genetic disease in which patients exhibit growth retardation, immunodeficiency, infertility, photosensitivity, and predisposition to cancer. The gene defective in BS has recently been cloned (named BLM) and was found to belong to an evolutionarily conserved helicase family, called RecQ. The recombinant BLMp protein has been shown to possess a helicase activity in vitro, suggesting that BS could be caused by a defect in a DNA metabolic reaction, such as replication or repair. Interestingly, BLM gene belongs to the helicase family, like the genes mutated in Werner Syndrome and Rothmund-Thomson syndrome (RTS). All three diseases have some common features, such as genetic instability and predisposition to cancer. But each disease has its own distinctive symptoms. For example, WS patients prematurely display many age-related features, including osteoporosis, atherosclerosis, diabetes and cataracts, which are not observed in BS or RTS. Also, WS individuals do not show immunodeficiency or photosensivity like BS patients. To understand the molecular mechanism of these human diseases, we propose to isolate the protein complexes containing each gene product. To investigate the mechanism of BS, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIa and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We find that BLM complexes isolated from a FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair. Much of this work has recently been published in Mol. Cell. Biol. ? In a paper published in EMBO J in 2005, we showed that BLAP75 is a component of all three BLM complexes from HeLa cells. Using siRNA knockdown techniques, we show that BLAP75 is essential for BLM complex stability in vivo. Consistent with a role in BLM-mediated processes, BLAP75 co-localizes with BLM in subnuclear foci in response to DNA damage, and its depletion impairs the recruitment of BLM to these foci. Depletion of BLAP75 by siRNA also results in deficient phosphorylation of BLM during mitosis, as well as defective cell proliferation. Moreover, cells depleted of BLAP75 display an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, BLAP75 is an essential component of the BLM-associated cellular machinery that maintains genome integrity. After our paper has been published, two other labs used genetic approaches to show that the yeast homolog of BLAP75 is also a component of RecQ helicase-Topo IIIa complex, and is required for maintaining genome stability. Thus, both biochemistry in human and genetics in yeast have reached the same conclusion. Together, these data suggest that BLAP75 and its homologs in various species have a conserved function in guarding genome.? ? It was shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase III (hTOPO III ), can process a toxic DNA intermediate generated in DNA repair reactions into a non-toxic product by a mechanism termed dissolution. In a collaboration with I. Hickson, G. Brown, and L. Li's labs, it was found that BLAP75 can strongly promote the dissolution catalyzed by hTOPO III by recruiting this enzyme to the toxic intermediate. This study demonstrates that BLM, hTOPO III and BLAP75 function as a molecular machine that maintains genome stability by efficiently processing the toxic intermediates generated during DNA repair. Identification of this machine and its biochemical activity should provide new means to screen drugs and could eventually contribute to the development of cancer therapies. This work was published this year in PNAS.? ? We are currently examining the importance of another BLM complex component, BLAP250, in genome maintanence. We have successfully knocked out this gene in chicken DT40 cells, and are investigating whether these cells have defects in the DNA repair.
Showing the most recent 10 out of 16 publications
