A positive involvement of RecQL4 in UV-induced S-phase arrest

RecQL4 belongs to a family of conserved RECQ helicases that are important in maintaining chromosomal integrity. Human patients lacking RecQL4 showed extreme sensitivity to UV and oxidation damage, suggesting that RecQL4 is involved in the damage signaling and/or repair. Here we show that human mutant cells lacking RecQL4 were defective in UV-induced S-phase arrest, whereas cells defective in bloom syndrome protein (BLM), another member of RecQ family exhibited a normal S-phase arrest following UV irradiation. In keeping with this, a targeted inhibition of RecQL4 expression in human 293 cells showed a defect in inducing S-phase (replication) arrest following UV treatment. Human mutant cells lacking RecQL4 protein were also defective in inducing S-phase arrest following hydroxyurea treatment. Together, our results suggest that RecQL4 may have a unique role in replication fork arrest, which may not be shared with other members of RecQ family such as BLM.     

Defending genome integrity during DNA replication: a proposed role for RecQ family helicases

The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in all organisms analysed to date. In human cells, representatives of this family include the proteins defective in the cancer predisposition disorder Bloom's syndrome and the premature ageing condition, Werner's syndrome. Several pieces of evidence suggest that RecQ family helicases form associations with one or more of the cellular topoisomerases, and together these heteromeric complexes manipulate DNA structure to effect efficient DNA replication, genetic recombination, or both. Here, we propose that RecQ helicases are required for ensuring that structural abnormalities arising during replication, such as at sites where replication forks encounter DNA lesions, are corrected with high fidelity. In mutants defective in these proteins, not only is replication abnormal, but cells display aberrant responses to DNA-damaging agents or inhibitors of DNA synthesis. We suggest that RecQ helicases may be important for the integration of cellular responses to these insults, such as by linking cell cycle checkpoint responses to recombinational repair. BioEssays 21:286–294, 1999. © 1999 John Wiley & Sons, Inc.     

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.     

RecQL4 cytoplasmic localization: Implications in mitochondrial DNA oxidative damage repair

RecQL4, one of the five human RecQ helicases, is crucial for genomic stability and RecQL4 when mutated leads to premature aging phenotypes in humans. Unlike other human RecQ helicases, RecQL4 is found both in the nucleus and the cytoplasm. While the nuclear localization signal (NLS) and the retention domain at the N-terminus are responsible for the nuclear localization of RecQL4, the signal for its cytoplasmic localization is essentially unknown. In this study, two functional nuclear exporting signals (NESs; pNES2 and pNES3) were identified at the C-terminus of RecQL4. Deletion of pNES2 drastically diminished the cytoplasmic localization of RecQL4. Strikingly, addition of ubiquitination tail at the C-terminus of RecQL4 substantially enriched the cytoplasmic fraction of RecQL4 only in the presence of functional pNES2. Immunofluorescence studies revealed that the cytoplasmic RecQL4 was localized in mitochondria. Consistent with its mitochondrial localization, a regulatory role for RecQL4 in the maintenance of mitochondrial DNA (mtDNA) copy number was demonstrated. Elevation of ectopic expression of RecQL4 increased the mtDNA copy number in HEK293 cells while RecQL4 knock down markedly decreased the mtDNA copy number in U2OS cells. Additionally, a substantially increased level of mitochondrial superoxide production, and a markedly decreased repair capacity for oxidative DNA damage were observed in the mitochondria of both RecQL4 deficient human fibroblasts and RecQL4-suppressed cancer cells. These data strongly suggest a regulatory role for RecQL4 in mitochondrial stability and function. Collectively, our study demonstrates that NES-mediated RecQL4 export to the cytoplasm is essential for the maintenance of mitochondrial genome stability.     

The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication

The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity. RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers. The human BLM and WRN helicases are required for normal S phase progression. In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild-type kinetics. To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1Delta cells. Unexpectedly, we find that these cells progress faster through S phase than their wild-type counterparts. Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase. However, completion of DNA replication is strongly retarded at the rDNA array of sgs1Delta cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus. These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase.     

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing,strand exchange and G4 DNA binding

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.     

Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability

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.     

RecQ helicases: multiple roles in genome maintenance

The RecQ helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. In humans, loss of RecQ helicase function is associated with predisposition to cancer and/or premature ageing. Recent data show that RecQ helicases have several roles during S phase of the cell cycle, ranging from facilitating the resumption of DNA synthesis at sites of replication fork breakdown to resolving structures during the process of homologous recombination.     

Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.     

Structure and Function of RecQ DNA Helicases

RecQ family helicases play important roles in coordinating genome maintenance pathways in living cells. In the absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including increased mitotic recombination, elevated chromosome missegregation, hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three of the five human RecQ family members give rise to genetic disorders associated with a predisposition to cancer and premature aging, highlighting the importance of RecQ proteins and their cellular activities for human health. Current evidence suggests that RecQ proteins act at multiple steps in DNA replication, including stabilization of replication forks and removal of DNA recombination intermediates, in order to maintain genome integrity. The cellular basis of RecQ helicase function may be explained through interactions with multiple components of the DNA replication and recombination machinery. This review focuses on biochemical and structural aspects of the RecQ helicases and how these features relate to their known cellular function, specifically in preventing excessive recombination.     

Structure and function of RecQ DNA helicases

RecQ family helicases play important roles in coordinating genome maintenance pathways in living cells. In the absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including increased mitotic recombination, elevated chromosome missegregation, hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three of the five human RecQ family members give rise to genetic disorders associated with a predisposition to cancer and premature aging, highlighting the importance of RecQ proteins and their cellular activities for human health. Current evidence suggests that RecQ proteins act at multiple steps in DNA replication, including stabilization of replication forks and removal of DNA recombination intermediates, in order to maintain genome integrity. The cellular basis of RecQ helicase function may be explained through interactions with multiple components of the DNA replication and recombination machinery. This review focuses on biochemical and structural aspects of the RecQ helicases and how these features relate to their known cellular function, specifically in preventing excessive recombination.     

RecQ4: the second replicative helicase?

Recent work has greatly contributed to the understanding of the biology and biochemistry of RecQ4. It plays an essential non-enzymatic role in the formation of the CMG complex, and thus replication initiation, by means of its Sld2 homologous domain. The helicase domain of RecQ4 has now been demonstrated to possess 3′–5′ DNA helicase activity, like the other members of the RecQ family. The biological purpose of this activity is still unclear, but helicase-dead mutants are unable to restore viability in the absence of wildtype RecQ4. This indicates that RecQ4 performs a second role, which requires helicase activity and is implicated in replication and DNA repair. Thus, it is clear that two helicases, RecQ4 and Mcm2-7, are integral to replication. The nature of the simultaneous involvement of these two helicases remains to be determined, and possible models will be proposed.     

The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins

BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions.     

RecQ family helicases in genome stability: Lessons from gene disruption studies in DT40 cells

Cells of all living organisms have evolved complex mechanisms to maintain genome stability. There is increasing evidence that spontaneous genomic instability occurs primarily during DNA replication. RecQ DNA helicases function during DNA replication and are essential for the maintenance of genome stability. In human cells, there exist five RecQ DNA helicases, and mutations of three of these helicases, encoded by the BLM, WRN and RECQL4 genes, give rise to the cancer predisposition disorders, Bloom syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS), respectively. Individuals suffering from WS and RTS also show premature aging phenotypes. Although the two remaining helicases, RECQL1 and RECQL5, have not yet been associated with heritable human diseases, a single nucleotide polymorphism of RECQL1 is associated with reduced survival of pancreatic cancer, and RecQl5 knockout mice show a predisposition to cancer. Here, we review the functions eukaryotic RecQ helicases, focusing primarily on BLM in the maintenance of genome stability through various pathways of nucleic acid metabolism and with special reference to DNA replication.     

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.     

Human diseases deficient in RecQ helicases

RecQ helicases are conserved from bacteria to man. Mutations in three of the human RecQ family members give rise to genetic disorders characterized by genomic instability and a predisposition to cancer. RecQ helicases are therefore caretakers of the genome, and although they do not directly regulate tumorigenesis, they influence stability and the rate of accumulation of genetic alterations, which in turn, result in tumorigenesis. Maintenance of genome stability by RecQ helicases likely involves their participation in DNA replication, recombination, and repair pathways.     

Drosophila homologue of the Rothmund-Thomson syndrome gene: essential function in DNA replication during development

Members of the RecQ family play critical roles in maintaining genome integrity. Mutations in human RecQL4 cause a rare genetic disorder, Rothmund-Thomson syndrome. Transgenic mice experiments showed that the RecQ4 null mutant causes embryonic lethality. Although biochemical evidence suggests that the Xenopus RecQ4 is required for the initiation of DNA replication in the oocyte extract, its biological functions during development remain to be elucidated. We present here our results in establishing the use of Drosophila as a model system to probe RecQ4 functions. Immunofluorescence experiments monitoring the cellular distribution of RecQ4 demonstrated that RecQ4 expression peaks during S phase, and RecQ4 is expressed only in tissues active in DNA replication, but not in quiescent cells. We have isolated Drosophila RecQ4 hypomorphic mutants, recq^EP and recq4²³, which specifically reduce chorion gene amplification of follicle cells by 4-5 fold, resulting in thin and fragile eggshells, and female sterility. Quantitative analysis on amplification defects over a 14-kb domain in chorion gene cluster suggests that RecQ4 may have a specific function at or near the origin of replication. A null allele recq4¹⁹ causes a failure in cell proliferation, decrease in DNA replication, chromosomal fragmentation, and lethality at the stage of first instar larvae. The mosaic analysis indicates that cell clones with homozygous recq4¹⁹ fail to proliferate. These results indicate that RecQ4 is essential for viability and fertility, and is required for most aspects of DNA replication during development.