Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein

RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1–α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.     

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.     

168 NMR study of RQC domain of BLM protein

BLM, a member of the RecQ helicase associated with the Bloom’s syndrome human genetic disorder, has been found to bind to noncanonical DNA with high affinity via its RecQ C-terminal domain (RQC). Using multi-dimensional NMR spectroscopy, we have determined the solution structure of BLM RQC, and found that BLM RQC retains the overall winged-helix motif previously observed for other RQC proteins. Comparison between BLM RQC and the RQC domain of its homologue, Werner syndrome protein (WRN RQC), revealed two major structural differences. Firstly, BLM RQC contains an extended 14-residue insertion forming a flexible loop between two first α-helices, only found in BLM RQC and not other RQC proteins. Secondly, in contrast to the third α-helix of WRN RQC, an unstructured loop was observed for this region of BLM RQC.     

Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase

There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.     

Modulation of Werner syndrome protein function by a single mutation in the conserved RecQ domain

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 site-directed 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.     

The HRDC domain of BLM is required for the dissolution of double Holliday junctions

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIalpha (TOPO IIIalpha) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.     

Stimulation of flap endonuclease-1 by the Bloom's syndrome protein

Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5'-flap endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.     

Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain

BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed.     

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases. Both WRN and BLM bind to the extreme C-terminal 18 amino acid tail of FEN-1 that is adjacent to the PCNA binding site of FEN-1. The importance of the WRN/BLM physical interaction with the FEN-1 C-terminal tail was confirmed by functional interaction studies with catalytically active purified recombinant FEN-1 deletion mutant proteins that lack either the WRN/BLM binding site or the PCNA interaction site. The distinct binding sites of WRN and PCNA and their combined effect on FEN-1 nuclease activity suggest that they may coordinately act with FEN-1. WRN was shown to facilitate FEN-1 binding to its preferred double-flap substrate through its protein interaction with the FEN-1 C-terminal binding site. WRN retained its ability to physically bind and stimulate acetylated FEN-1 cleavage activity to the same extent as unacetylated FEN-1. These studies provide new insights to the interaction of WRN and BLM helicases with FEN-1, and how these interactions might be regulated with the PCNA–FEN-1 interaction during DNA replication and repair.     

The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA

The product of the gene mutated in Bloom's syndrome, BLM, is a 3′–5′ DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg²⁺, is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.     

The Human RecQ helicases, BLM and RECQ1, display distinct DNA substrate specificities

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3'-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.     

Characterization and mutational analysis of the RecQ core of the bloom syndrome protein

Bloom syndrome protein forms an oligomeric ring structure and belongs to a group of DNA helicases showing extensive homology to the Escherichia coli DNA helicase RecQ, a suppressor of illegitimate recombination. After over-production in E.coli, we have purified the RecQ core of BLM consisting of the DEAH, RecQ-Ct and HRDC domains (amino acid residues 642-1290). The BLM(642-1290) fragment could function as a DNA-stimulated ATPase and as a DNA helicase, displaying the same substrate specificity as the full-size protein. Gel-filtration experiments revealed that BLM(642-1290) exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg(2+) and ATPgammaS. Rates of ATP hydrolysis and DNA unwinding by BLM(642-1290) showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a lambda Spi(-) assay, we have found that the BLM(642-1290) fragment is able to partially substitute for the RecQ helicase in suppressing illegitimate recombination in E.coli. A deletion of 182 C-terminal amino acid residues of BLM(642-1290), including the HRDC domain, resulted in helicase and single-stranded DNA-binding defects, whereas kinetic parameters for ATP hydrolysis of this mutant were close to the BLM(642-1290) values. This confirms the prediction that the HRDC domain serves as an auxiliary DNA-binding domain. Mutations at several conserved residues within the RecQ-Ct domain of BLM reduced ATPase and helicase activities severely as well as single-stranded DNA-binding of the enzyme. Together, these data define a minimal helicase domain of BLM and demonstrate its ability to act as a suppressor of illegitimate recombination.     

G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition

To understand the specific genetic instabilities associated with deficiencies in RecQ family helicases, we have studied the substrate preferences of two closely related members of this family, human BLM and Saccharomyces cerevisiae Sgs1p. Here we show that both BLM and Sgs1p preferentially unwind G4 DNA relative to Holliday junction substrates, and that substrate preference reflects binding affinity and maps to the conserved central helicase domain. We identify the porphyrin N-methyl mesoporphyrin IX (NMM) as a specific inhibitor of G4 DNA unwinding, and show that in the presence of NMM the helicase becomes trapped on the NMM–G4 DNA complex, consuming ATP but unable to unwind or dissociate. These results suggest that BLM and Sgs1p function proactively in replication to remove G4 DNA structures which would otherwise present obstacles to fork progression, rather than by promoting recombination to restart a fork that has stalled.     

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.     

Identification of the SSB Binding Site on E. coli RecQ Reveals a Conserved Surface for Binding SSB's C Terminus

RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities, including recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding protein (SSB) stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain, which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares a striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli exonuclease I, although the SSB binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the winged-helix domain in helicase activity beyond SSB protein binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.     

Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes

Bloom''s syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM–DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.     

Werner syndrome protein directly binds to the AAA ATPase p97/VCP in an ATP-dependent fashion

We have previously shown that the Werner syndrome helicase, WRNp, a member of the RecQ helicase family, forms a tight molecular complex with the p97/Valosin containing protein (VCP), a member of the AAA (ATPases associated with diverse cellular activities) family of proteins. This interaction is disrupted by chemical agents that confer DNA damage, suggesting that VCP plays an important role in the signal-dependent release of WRNp from its nucleolar sequestration site. Here, we characterized the structural requirements for interactions between WRNp and VCP and for the nuclear localization of VCP. We discovered that VCP directly binds to the RQC (RecQ conserved) domain of WRNp, which is a highly conserved motif common to the RecQ helicase family. This interaction is ATP-dependent, suggesting that VCP plays a mechanistic role in releasing WRNp from the nucleolus. Immunohistochemical analysis of various VCP domains and mutated proteins expressed in vitro demonstrated that VCP may contain several hierarchical cellular localization motifs within its domain structure.     

A Nucleotide-dependent and HRDC Domain-dependent Structural Transition in DNA-bound RecQ Helicase

The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF₄. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.     

Mechanism of RecQ helicase mechanoenzymatic coupling reveals that the DNA interactions of the ADP-bound enzyme control translocation run terminations

The processing of various DNA structures by RecQ helicases is crucial for genome maintenance in both bacteria and eukaryotes. RecQ helicases perform active destabilization of DNA duplexes, based on tight coupling of their ATPase activity to moderately processive translocation along DNA strands. Here, we determined the ATPase kinetic mechanism of E. coli RecQ helicase to reveal how mechanoenzymatic coupling is achieved. We found that the interaction of RecQ with DNA results in a drastic acceleration of the rate-limiting ATP cleavage step, which occurs productively due to subsequent rapid phosphate release. ADP release is not rate-limiting and ADP-bound RecQ molecules make up a small fraction during single-stranded DNA translocation. However, the relatively rapid release of the ADP-bound enzyme from DNA causes the majority of translocation run terminations (i.e. detachment from the DNA track). Thus, the DNA interactions of ADP-bound RecQ helicase, probably dependent on DNA structure, will mainly determine translocation processivity and may control the outcome of DNA processing. Comparison with human Bloom''s syndrome (BLM) helicase reveals that similar macroscopic parameters are achieved by markedly different underlying mechanisms of RecQ homologs, suggesting diversity in enzymatic tuning.     

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.