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.     

Analysis of helicase activity and substrate specificity of Drosophila RECQ5

RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3′ Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5′ Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM.     

SUVi and BACH1: a new subfamily of mammalian helicases?

The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in prokaryotes and eukaryotes. Members of this family are genes responsible for cancer predisposition disorders like Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome. Here, we show the sequence homologies between two recently identified mammalian helicases, namely SUVi and BACH1. These two genes also share strong homologies with other members of the RecQ family. On the basis of published data and sequence analysis we suggest that SUVi/BACH1 may represent a novel subfamily of mammalian helicases, functioning in the processing of lesions induced by different genotoxic agents.     

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.     

A conserved G4 DNA binding domain in RecQ family helicases

RecQ family helicases play important roles at G-rich domains of the genome, including the telomeres, rDNA, and immunoglobulin switch regions. This appears to reflect the unusual ability of enzymes in this family to unwind G4 DNA. How RecQ family helicases recognize this substrate has not been established. Here, we show that G4 DNA is a preferred target for BLM helicase within the context of long DNA molecules. We identify the RQC domain, found only in RecQ family enzymes, as an independent, high affinity and conserved G4 DNA binding domain; and show that binding to Holliday junctions involves both the RQC and the HRDC domains. These results provide mechanistic understanding of differences and redundancies of function and activities among RecQ family helicases, and of how deficiencies in human members of this family may contribute to genomic instability and disease.     

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.     

大肠杆菌RecQ解旋酶的表达纯化和生物学活性研究

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达,并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性. 体外诱导表达获得纯度达90% 以上并具有高活性的大肠杆菌重组RecQ解旋酶,其可溶性好;经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性. 较之双链DNA（dsDNA）,大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合( P<0.01 ),但与长度不同的dsDNA的结合特性有差异(P<0.01)而与ssDNA没有差异(P>0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P<0.05);大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P<0.01). 这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制,并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助.     

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.     

RECQ1 helicase interacts with human mismatch repair factors that regulate genetic recombination

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.     

RecQ helicases: guardian angels of the DNA replication fork

Since the original observations made in James German’s Laboratory that Bloom’s syndrome cells lacking BLM exhibit a decreased rate of both DNA chain elongation and maturation of replication intermediates, a large body of evidence has supported the idea that BLM, and other members of the RecQ helicase family to which BLM belongs, play important roles in DNA replication. More recent evidence indicates roles for RecQ helicases in what can broadly be defined as replication fork ‘repair’ processes when, for example, forks encounter lesions or adducts in the template, or when forks stall due to lack of nucleotide precursors. More specifically, several roles in repair of damaged forks via homologous recombination pathways have been proposed. RecQ helicases are generally only recruited to sites of DNA replication following fork stalling or disruption, and they do so in a checkpoint-dependent manner. There, in addition to repair functions, they aid the stabilisation of stalled replication complexes and seem to contribute to the generation and/or transduction of signals that enforce S-phase checkpoints. RecQ helicases also interact physically and functionally with several key players in DNA replication, including RPA, PCNA, FEN1 and DNA polymerase δ. In this paper, we review the evidence that RecQ helicases contribute to the impressively high level of fidelity with which genome duplication is effected.     

RECQ1 possesses DNA branch migration activity

RecQ helicases are essential for the maintenance of genome stability. Five members of the RecQ family have been found in humans, including RECQ1, RECQ5, BLM, WRN, and RECQ4; the last three are associated with human diseases. At this time, only BLM and WRN helicases have been extensively characterized, and the information on the other RecQ helicases has only started to emerge. Our current paper is focused on the biochemical properties of human RECQ1 helicase. Recent cellular studies have shown that RECQ1 may participate in DNA repair and homologous recombination, but the exact mechanisms of how RECQ1 performs its cellular functions remain largely unknown. Whereas RECQ1 possesses poor helicase activity, we found here that the enzyme efficiently promotes DNA branch migration. Further analysis revealed that RECQ1 catalyzes unidirectional three-stranded branch migration with a 3' --> 5' polarity. We show that this RECQ1 activity is instrumental in specific disruption of joint molecules (D-loops) formed by a 5' single-stranded DNA invading strand, which may represent dead end intermediates of homologous recombination in vivo. The newly found enzymatic properties of the RECQ1 helicase may have important implications for the function of RECQ1 in maintenance of genomic stability.     

RecQL4: a helicase linking formation and maintenance of a replication fork

RecQ family helicases are conserved from bacteria to human. Across the species, they are known to be required for protecting genome from various genotoxic stresses. In human, five RecQ-related helicases have been identified and three of them, BLM, WRN and RecQL4, have been shown to be responsible for genetic disorders, Bloom, Werner and Rothmund-Thomson syndrome, respectively, which are characterized by cancer predisposition and premature ageing. RecQL4, the N-terminal portion of which shares similarity with Sld2 known to be required for assembly of a replication complex in yeasts, is unique in that it has been shown to be essential for the initiation phase of normal DNA replication. Recent biochemical characterization demonstrated the 3'-5' DNA helicase activity associated with RecQL4. Understanding the molecular basis for how RecQ helicases are involved in generation and maintenance of normal and stalled DNA replication forks would be crucial to elucidation of the mechanisms of replication initiation as well as to that of how the loss of these conserved helicases leads to varieties of disease phenotypes.     

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.     

RecQ helicases in DNA double strand break repair and telomere maintenance

Organisms are constantly exposed to various environmental insults which could adversely affect the stability of their genome. To protect their genomes against the harmful effect of these environmental insults, organisms have evolved highly diverse and efficient repair mechanisms. Defective DNA repair processes can lead to various kinds of chromosomal and developmental abnormalities. RecQ helicases are a family of evolutionarily conserved, DNA unwinding proteins which are actively engaged in various DNA metabolic processes, telomere maintenance and genome stability. Bacteria and lower eukaryotes, like yeast, have only one RecQ homolog, whereas higher eukaryotes including humans possess multiple RecQ helicases. These multiple RecQ helicases have redundant and/or non-redundant functions depending on the types of DNA damage and DNA repair pathways. Humans have five different RecQ helicases and defects in three of them cause autosomal recessive diseases leading to various kinds of cancer predisposition and/or aging phenotypes. Emerging evidence also suggests that the RecQ helicases have important roles in telomere maintenance. This review mainly focuses on recent knowledge about the roles of RecQ helicases in DNA double strand break repair and telomere maintenance which are important in preserving genome integrity.     

Substrate-specific inhibition of RecQ helicase

The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (K_i) for NMM inhibition of G4 DNA unwinding is 1.7 µM, approximately two orders of magnitude below the K_i for inhibition of duplex DNA unwinding (>100 µM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.     

Unique and important consequences of RECQ1 deficiency in mammalian cells

Five members of the RecQ subfamily of DEx-H-containing DNA helicases have been identified in both human and mouse, and mutations in BLM, WRN, and RECQ4 are associated with human diseases of premature aging, cancer, and chromosomal instability. Although a genetic disease has not been linked to RECQ1 mutations, RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage.(1) Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous γ-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks.(2) Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin.(2) RECQ1 catalytic activities(3) and interactions with DNA repair proteins(2,4,5) are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis.     

Rising from the RecQ-age: the role of human RecQ helicases in genome maintenance

The RecQ helicases are guardians of the genome. Members of this conserved family of proteins have a key role in protecting and stabilizing the genome against deleterious changes. Deficiencies in RecQ helicases can lead to high levels of genomic instability and, in humans, to premature aging and increased susceptibility to cancer. Their diverse roles in DNA metabolism, which include a role in telomere maintenance, reflect interactions with multiple cellular proteins, some of which are multifunctional and also have very diverse functions. The results of in vitro cellular and biochemical studies have been complimented by recent in vivo studies using genetically modified mouse strains. Together, these approaches are helping to unravel the mechanism(s) of action and biological functions of the RecQ helicases.     

Cloning of Two New Human Helicase Genes of the RecQ Family: Biological Significance of Multiple Species in Higher Eukaryotes

Two new human DNA helicase genes,RecQ4andRecQ5,that belong to the RecQ helicase family were cloned and characterized. The addition of these genes increases the total to five helicase genes in the human RecQ family, which includes helicases involved in Bloom and Werner syndromes, the genetic diseases manifesting the distinctive but overlapping clinical phenotypes of immunodeficiency, premature aging, and an enhanced risk of cancer. The RecQ4 helicase is as large as the Bloom (BLM) and Werner (WRN) helicases, and its gene expression profile is organ-specific, resembling that of BLM helicase. In contrast, the RecQ5 helicase has a low molecular weight, similar to the human progenitor RecQ1 helicase, and is expressed in all the organs examined. All five human helicase genes are expressed in cultured K562 leukemia and fibroblast cells. Synchronized K562 cell cultures showed that the genesRecQ4andBLM,andRecQ1andWRN,seem to be upregulated at the G1/S and G2/M phases, respectively, of the cell cycle. The biological significance of multiple species of human RecQ helicases, which are apparently nonessential for life but may be related to distinct diseases, is discussed in light of the fact that unicellular organisms, likeEscherichia coliand yeast, contain only one species of helicase of this particular family.     

RPA alleviates the inhibitory effect of vinylphosphonate internucleotide linkages on DNA unwinding by BLM and WRN helicases

Bloom (BLM) and Werner (WRN) syndrome proteins are members of the RecQ family of SF2 DNA helicases. In this paper, we show that restricting the rotational DNA backbone flexibility, by introducing vinylphosphonate internucleotide linkages in the translocating DNA strand, inhibits efficient duplex unwinding by these enzymes. The human single-stranded DNA binding protein replication protein A (RPA) fully restores the unwinding activity of BLM and WRN on vinylphosphonate-containing substrates while the heterologous single-stranded DNA binding protein from Escherichia coli (SSB) restores the activity only partially. Both RPA and SSB fail to restore the unwinding activity of the SF1 PcrA helicase on modified substrates, implying specific interactions of RPA with the BLM and WRN helicases. Our data highlight subtle differences between SF1 and SF2 helicases and suggest that although RecQ helicases belong to the SF2 family, they are mechanistically more similar to the SF1 PcrA helicase than to other SF2 helicases that are not affected by vinylphosphonate modifications.