The Bloom's Syndrome Protein (BLM) Interacts with MLH1 but Is Not Required for DNA Mismatch Repair

Abstract

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre-and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.Bloom's syndrome (BS) 1 is a rare autosomal recessive disorder characterized by immunodeficiency, short stature, male infertility, and an increased risk of a broad spectrum of cancers (1). Cells isolated from BS individuals are characterized by cytogenetic abnormalities, with the hallmark feature of hyperrecombination between sister chromatids. BS chromosomes also display increased levels of breaks, translocations, quadriradial formations, and telomeric associations (2).The gene mutated in BS was positionally cloned and named BLM; it encodes a 1417-amino acid protein with strong homology to the Escherichia coli RecQ family of DNA and RNA helicases (3). The E. coli RecQ helicase participates in homologous recombination and suppresses illegitimate recombination (4, 5). Other eukaryotic RecQ family members include Sgs1p from Saccharomyces cerevisiae and Rqh1p from Schizosaccharomyces pombe; loss of function of either of these helicases results in genomic instability (6, 7). Mutations in other human RecQ helicases result in the rare autosomal recessive disorders Werner's syndrome and Rothmund-Thomson syndrome, also characterized by chromosomal instability and cancer predisposition (8, 9).The BLM helicase unwinds duplex DNA from 3Ј to 5Ј in the presence of ATP (10, 11). It also selectively recognizes and promotes branch migration of Holliday junctions in vitro (12). BLM can be found in a large protein complex in the nucleus with other proteins involved in DNA repair such as BRCA1, ATM, MLH1, MSH2, MSH6, and replication factor C (13). However, direct interactions of BLM have only been demonstrated biochemically with replication protein A (RPA) and topoisomerase III␣ (14 -16). These experiments suggest that the BLM helicase interacts with a variety of nuclear proteins to perform functions in DNA replication, recombination, or repair.To understand the role of the BLM helicase in maintaining genomic stability, a yeast two-hybrid screen was used to identify...

Molecular Mechanisms of Mismatch Repair Genes in Cancer A Brief Review

“…The human Bloom syndrome helicase (BLM), a component of BASC, was shown to directly interact with MLH1 [71]. It has been hypothesized that MSH4-MSH5 and MLH1-MLH3 complexes promote the formation and stabilization of Holliday junctions and their preferential resolution into cross-overs rather than non cross-overs [72].…”

The mismatch DNA repair heterodimer, hMSH2/6, regulates BLM helicase

“…In mammalian cells, about half of all DNA DSB are repaired by mechanisms involving HRR (Liang et al, 1998). As cell extracts lacking functional BLM helicase activity are competent for DNA MMR (Langland et al, 2001), the interaction of BLM with mismatch proteins may be significant for specific DNA events, for example, HRR, independent of the repair of single DNA mismatches. Studies using a yeast model system suggest that MSH2 and the SGS1 helicase play a significant role in HRR (Marsischky et al, 1999;Evans et al, 2000;Frei and Gasser, 2000;Myung et al, 2001).…”

“…BLM associates with other proteins involved in DNA repair such as BRCA1, ATM, hMLH1, hMSH2, hMSH6, and replication factor C to form a complex called BASC that was isolated from p53-deficient HeLa cells (Wang et al, 2000). BLM binds directly to hMLH1, but is not required for DNA MMR (Langland et al, 2001;Pedrazzi et al, 2001). The Saccharomyces cerevisiae homolog of BLM, SGS1, suppresses genomic instability and homologous recombination (Myung et al, 2001).…”

The Bloom's Syndrome Helicase Interacts Directly with the Human DNA Mismatch Repair Protein hMSH6

“…Since BS cells are not deficient in MMR, it has been proposed that the hMLH1 interaction with BLM may play a role in HR (Langland et al, 2001;Pedrazzi et al, 2001). In this study, we set out to test whether BLM interacts also with other components of the MMR system.…”

“…We and others have recently demonstrated that BLM interacts directly with the MMR protein hMLH1 (Langland et al, 2001;Pedrazzi et al, 2001). Since BS cells are not deficient in MMR, it has been proposed that the hMLH1 interaction with BLM may play a role in HR (Langland et al, 2001;Pedrazzi et al, 2001).…”