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.

Cutaneous eruptions are a commonly reported adverse drug reaction. Cutaneous adverse drug reactions in the pediatric population have a significant impact on patients' current and future care options. A patient's recollection of having a "rash" when they took a medication as a child is a frequent reason for not prescribing a particular treatment. The quick detection and treatment of cutaneous adverse drug reactions, plus identification of the causative agent, are essential for preventing the progression of the reaction, preventing additional exposures, and ensuring the appropriate use of medications for both the current condition and others as the patient ages. The purpose of this review is to discuss a reasonable approach to recognition and initial management of cutaneous adverse drug reactions in children.

The BRCA1 associated C-terminal helicase (BACH1) associated with breast cancer has been implicated in double strand break (DSB) repair. More recently, BACH1 (FANCJ) has been genetically linked to the chromosomal instability disorder Fanconi Anemia (FA). Understanding the roles of BACH1 in cellular DNA metabolism and how BACH1 dysfunction leads to tumorigenesis requires a comprehensive investigation of its catalytic mechanism and molecular functions in DNA repair. In this study, we have determined that BACH1 helicase contacts with both the translocating and the non-translocating strands of the duplex are critical for its ability to track along the sugar phosphate backbone and unwind dsDNA. An increased motor ATPase of a BACH1 helicase domain variant (M299I) enabled the helicase to unwind the backbone-modified DNA substrate in a more proficient manner. Alternatively, increasing the length of the 5′ tail of the DNA substrate allowed BACH1 to overcome the backbone discontinuity, suggesting that BACH1 loading mechanism is critical for its ability to unwind damaged DNA molecules.

Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using sitedirected mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures. Werner syndrome (WS)2 is an autosomal recessive disorder characterized by premature aging after puberty with multiple symptoms, including graying and loss of hair, bilateral cataracts, atherosclerosis, diabetes mellitus, osteoporosis, hypogonadism, and cancer predisposition (1). In addition, WS cells exhibit genomic instability (2), replication defects (3), aberrant telomere maintenance (4), and a gene expression profile that resembles that of normal human aging (5). The WRN gene (also known as RECQ3) located at chromosome 8p11-12, encodes a nuclear ϳ162-kDa protein (WRN), which exhibits a DNA-dependent ATPase, DNA helicase activity on double strand DNA (dsDNA) with 3Ј 3 5Ј-polarity (6), and a functional N-terminal 3Ј 3 5Ј-exonuclease activity (7).WRN belongs to a family of RecQ helicases that also includes RECQ1 (8, 9), RECQ2 (10), RECQ4 (11), and RECQ5 (11). Functions of RecQ helicases based on cellular and biochemical evidence suggest their involvement in DNA replication, DNA repair, recombination, and telomere maintenance. Clinically, defects in certain RECQ genes have been linked to rare genetic disorders, including Bloom syndrome (RECQ2), Werner syndrome (RECQ3), and Rothmund-Thompson syndrome (RECQ4). One of the prominent characteristics common to all RecQ helicases is the sequence conservation of the seven helicase motifs centrally located in the respective proteins (12). In addition to the helicase domain, a majority of RecQ helicases shares another conserved sequence designated the RecQ C-terminal (RQC) region (13). The WRN RQC region is located between amino acid (aa) residues 872 and 1045 and is homologous to the part of the catalytic core of Escherichia coli RecQ for which the three-dimensional structure has revealed the presence of a winged helix-turn-helix (wHTH) motif (14). Moreover, of the 17 WRN-interacting proteins, 14 that are functionally signifi...

DNA helicases have essential roles in nucleic acid metabolism by facilitating cellular processes including replication, recombination, DNA repair, and transcription. The vital roles of helicases in these pathways are reflected by their emerging importance in the maintenance of genomic stability. Recently, a number of human diseases with cancer predisposition have been shown to be genetically linked to a specific helicase defect. This has led researchers to further investigate the roles of helicases in cancer biology, and to study the efficacy of targeting human DNA helicases for anti-cancer drug treatment. Helicase-specific inhibition in malignant cells may compromise the high proliferation rates of cancerous tissues. The role of RecQ helicases in response to replicational stress suggests a molecular target for selectively eliminating malignant tumor cells by a cancer chemotherapeutic agent. Alternate DNA secondary structures such as G-quadruplexes that may form in regulatory regions of oncogenes or G-rich telomere sequences are potential targets for cancer therapy since these sequence-specific structures are proposed to affect gene expression and telomerase activation, respectively. Small molecule inhibitors of G-quadruplex helicases may be used to regulate cell cycle progression by modulating promotor activation or disrupting telomere maintenance, important processes of cellular transformation. The design of small molecules which deter helicase function at telomeres may provide a molecular target since telomerase activity is necessary for the proliferation of numerous immortal cells. Although evidence suggests that helicases are specifically inhibited by certain DNA binding compounds, another area of promise in anti-cancer therapy is siRNA technology. Specific knockdown of helicase expression can be utilized as a means to sensitize oncogenic proliferating cell lines. This review will address these topics in detail and summarize the current avenues of research in anti-cancer therapy targeting helicases through small molecule inhibitors of DNA-protein complexes, DNA binding drugs, or down-regulation of helicase gene expression.