Helicases are molecular motor proteins that couple the hydrolysis of NTP to nucleic acid unwinding. The growing number of DNA helicases implicated in human disease suggests that their vital specialized roles in cellular pathways are important for the maintenance of genome stability. In particular, mutations in genes of the RecQ family of DNA helicases result in chromosomal instability diseases of premature aging and/or cancer predisposition. We will discuss the mechanisms of RecQ helicases in pathways of DNA metabolism. A review of RecQ helicases from bacteria to human reveals their importance in genomic stability by their participation with other proteins to resolve DNA replication and recombination intermediates. In the light of their known catalytic activities and protein interactions, proposed models for RecQ function will be summarized with an emphasis on how this distinct class of enzymes functions in chromosomal stability maintenance and prevention of human disease and cancer.Key words: aging, cancer, DNA repair, genomic instability, helicase, RecQ. INTRODUCTIONUnderstanding the molecular mechanisms of RecQ helicases is fundamental to deciphering the roles of these enzymes in cellular DNA metabolism. Since the discovery of Escherichia coli RecQ and its implicated role in genetic recombination, the world of RecQ helicases has become significantly more complex with the identification and characterization of a number of eukaryotic RecQ helicases that are important in the replicational stress response and maintenance of genomic stability.Classification of RecQ helicases and molecular-genetic analyses of their biochemical and cellular functions gained prominence with the understanding that certain rare genetic disorders [WS (Werner syndrome), BS (Bloom syndrome) and RTS (Rothmund-Thomson syndrome)] are a consequence of mutations in the human RecQ genes WRN, BLM and RECQ4 respectively. WS is characterized by premature aging with an elevated risk of age-associated diseases such as cancer, atherosclerotic cardiovascular disease, diabetes mellitus (Type II) and osteoporosis [1]. Epigenetic inactivation of WRN is detected in a number of human cancers [2]. BS is associated with a very high incidence of different types of cancers, both solid tumours and leukaemia, and also manifested by skin disorders, proportional dwarfism, immunodeficiency and male sterility [1]. People with RTS, also known as poikiloderma congenitale, displays growth deficiency, photosensitivity with poikilodermatous skin changes, early greying and hair loss, juvenile cataracts and a predisposition to malignancy, especially osteogenic sarcomas [1]. Apart from RTS, RECQ4 mutations were detected in Finnish patients with an autosomal recessive disorder RAPADILINO syndrome [radial hypoplasia/aplasia, patellae hypoplasia/aplasia and cleft or highly arched palate, diarrhoea and dislocated joints, little size (height at least 2 S.D. smaller than the average height) and limb malformation, nose slender and normal intelligence] [3]. Although many features o...

The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN H-R ) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN H-R helicase activity was significantly reduced on RPAdependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN H-R had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPAdependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM 642-1290 , that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.Within the last decade, several genetic disorders with premature aging and/or cancer have been identified in which a gene member of the RecQ helicase family is mutated (1, 2). RecQ helicases share a centrally located domain of ϳ450 residues that contains the seven conserved helicase motifs (for review, see Ref.3). The founding member of the RecQ family, Escherichia coli RecQ helicase, has been extensively studied biochemically and has been genetically implicated in DNA recombination. A single yeast RecQ helicase, Sgs1 or Rqh1, is found in the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, respectively, and these helicases are thought to be important in the cellular response to DNA-damaging agents and maintenance of genome stability. RecQ helicases have also been identified in a number of higher eukaryotes, including Xenopus laevis (focus forming activity 1 (FFA-1) 1 ), Drosophila melanogaster (DmBLM and DmRecQ5), and Caenorhabditis elegans (WRN-1, Ce-RCQ5, HIM-6, and RECQL/Q1). These helicases have proposed functions in ...

RecQ helicases are required for the maintenance of genome stability. Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function. In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1. Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 +/- 0.6 nm, suggesting that RECQ1 forms dimers in solution. The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt. We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA). The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA. However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1. In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.

Understanding the molecular and cellular functions of RecQ helicases has attracted considerable interest since several human diseases characterized by premature aging and/or cancer have been genetically linked to mutations in genes of the RecQ family. Although a human disease has not yet been genetically linked to a mutation in RECQ1, the prominent roles of RecQ helicases in the maintenance of genome stability suggest that RECQ1 helicase is likely to be important in vivo. To acquire a better understanding of RECQ1 cellular and molecular functions, we have investigated its protein interactions. Using a co-immunoprecipitation approach, we have identified several DNA repair factors that are associated with RECQ1 in vivo. Direct physical interaction of these repair factors with RECQ1 was confirmed with purified recombinant proteins. Importantly, RECQ1 stimulates the incision activity of human exonuclease 1 and the mismatch repair recognition complex MSH2/6 stimulates RECQ1 helicase activity. These protein interactions suggest a role of RECQ1 in a pathway involving mismatch repair factors. Regulation of genetic recombination, a proposed role for RecQ helicases, is supported by the identified RECQ1 protein interactions and is discussed.