Helicase And RNaseD C-terminal Domain Research Articles

The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3'-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5'-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling.

DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom’s syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

Domain Architecture of RecQ Helicase Defines Mechanochemical Linkage via Multipartite Interactions with DNA Substrate during Unwinding Activity

RecQ helicases are ubiquitous enzymes that safeguard the genome by playing multiple roles in DNA repair, replication and recombination. They are unique in their capability to process a wide range of non-canonical structures associated with DNA metabolic intermediates. DNA-binding domains linked to the RecQ helicase core, including the winged-helix (WHD) and helicase-and-RnaseD-C-terminal (HRDC) domains, are thought to confer substrate specificity and modulate enzymatic activities. We combined ensemble biophysical and single-molecule magnetic tweezers assays to determine the mechanochemical linkage between ATP hydrolytic and DNA-restructuring activities of E. coli RecQ constructs of varying domain architecture. We compared the activities of wild-type, HRDC point mutant and deletion mutant, and WHD-HRDC deletion mutant constructs using several defined DNA structures and experimental geometries. We show that the WHD enhances unwinding processivity by stabilizing enzyme-DNA interactions, whereas the HRDC domain increases the overall DNA affinity but hinders the unwinding and ATPase activities of the helicase through interactions with single-stranded (ss) DNA regions. Intriguingly, HRDC-ssDNA interactions stabilize the pausing of the helicase during unwinding of a DNA hairpin in which both nascent ssDNA strands are mechanically strained, but not that of gapped duplex DNA in which one of the liberated ssDNA strands is mechanically unconstrained. Our study reveals that both the DNA substrate geometry and the contribution of auxiliary DNA-binding domains greatly influence the mechanochemical behavior of the helicase and the outcome of DNA-processing reactions, bearing consequences on the diverse in vivo actions of RecQ enzymes.

Solution Small Angle X-ray Scattering (SAXS) Studies of RecQ from Deinococcus radiodurans and Its Complexes with Junction DNA Substrates

RecQ helicases, essential enzymes for maintaining genome integrity, possess the capability to participate in a wide variety of DNA metabolisms. They can initiate the homologous recombination repair pathway by unwinding damaged dsDNA and suppress hyper-recombination by promoting Holliday junction (HJ) migration. To learn how DrRecQ participates in the homologous recombination repair pathway, solution structures of Deinococcus radiodurans RecQ (DrRecQ) and its complexes with DNA substrates were investigated by small angle x-ray scattering. We found that the catalytic core and the most N-terminal HRDC (helicase and RNase D C-terminal) domain (HRDC1) undergo a conformational change to a compact state upon binding to a junction DNA. Furthermore, models of DrRecQ in complexes with two kinds of junction DNA (fork junction and HJ) were built based on the small angle x-ray scattering data, and together with the EMSA results, possible binding sites were proposed. It is demonstrated that two DrRecQ molecules bind to the opposite arms of HJ. This architecture is similar to the RuvAB complex and is hypothesized to be highly conserved in the other HJ migration proteins. This work provides us new clues to understand the roles DrRecQ plays in the RecFOR pathway.

Recruitment of HRDC domain of WRN and BLM to the sites of DNA damage induced by mitomycin C and methyl methanesulfonate

The HRDC (helicase and RNase D C-terminal) domain at the C-terminal of WRNp (Werner protein) (1150-1229 amino acids) and BLMp (Bloom protein) (1212-1292 amino acids) recognize laser microirradiation-induced DNA dsbs (double-strand breaks). However, their role in the recognition of DNA damage other than dsbs has not been reported. In this work, we show that HRDC domain of both the proteins can be recruited to the DNA damage induced by MMS (methyl methanesulfonate) and MMC (methyl mitomycin C). GFP (green fluorescent protein)-tagged HRDC domain produces distinct foci-like respective wild-types after DNA damage induced by the said agents and co-localize with γ-H2AX. However, in time course experiment, we observed that the foci of HRDC domain exist after 24 h of removal of the damaging agents, while the foci of full-length protein disappear completely. This indicates that the repair events are not completed by the presence of protein corresponding to only the HRDC domain. Consequently, cells overexpressing the HRDC domain fail to survive after DNA damage, as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Moreover, 24 h after removal of damaging agents, the extent of DNA damage is greater in cells overexpressing HRDC domain compared with corresponding wild-types, as observed by comet assay. Thus, our observations suggest that HRDC domain of both WRN and BLM can also recognize different types of DNA damages, but for the successful repair they fail to respond to subsequent repair events.

Two ReCA Domains Comprise a Minimal Functional Unit of the Human BlM Helicase

Human Bloom's syndrome helicase (BLM), a member of the RecQ family, plays key roles in homologous recombination (HR) based DNA repair mechanisms. BLM is a multidomain protein comprising four major domains. i, The large N-terminal domain is responsible for oligomerization of the helicase, and for maintaining protein-protein interactions required for double Holliday-junction (DHJ) dissolution and D-loop disruption. ii, The core of BLM consists of two conserved RecA domains that form the ATP-binding cleft and bind unwound DNA segments. iii, The conserved RecQ-C-terminal domain (RQC) is located downstream of the RecA domains. This domain contains the Zn2+-binding region which is essential for correct folding. iv, The C-terminal HRDC (helicase- and RNase-D C-terminal) domain is indispensable for DHJ dissolution. In a quest for the minimal functional helicase core of BLM, we produced a series of truncated constructs and tested them for ssDNA binding and translocation, duplex unwinding, and clearance of hRad51 recombinase-nucleoprotein filaments. Surprisingly we found that the deletion of the N-terminal and HRDC domains (BLM642-1077) did not change the activity profile of BLM compared with the widely used RecQ-homolog construct (BLM642-1290), and the strand annealing activity was also retained. Moreover, a construct comprising only the two RecA domains (BLM642-1005), thus lacking even the RQC domain, unwound splayed-arm double stranded (ds) DNA substrate and displayed hRad51-clearance activity. However, this construct exhibited no ssDNA-dependent enzymatic activation, indicating no ssDNA translocation. In summary, these results suggest that the ‘helicase core’ of DEAH-box BLM works similarly to the DEAD-box RNA helicases (Vasa, eIF4A) in which the two RecA domains are capable of disrupting short dsRNA regions and nucleoprotein filaments with no processive translocation. In this sense, additional domains of BLM display modulatory partner-like functions increasing processivity and providing specific activities.

Structure and function of the regulatory HRDC domain from human Bloom syndrome protein.

The helicase and RNaseD C-terminal (HRDC) domain, conserved among members of the RecQ helicase family, regulates helicase activity by virtue of variations in its surface residues. The HRDC domain of Bloom syndrome protein (BLM) is known as a critical determinant of the dissolution function of double Holliday junctions by the BLM–Topoisomerase IIIα complex. In this study, we determined the solution structure of the human BLM HRDC domain and characterized its DNA-binding activity. The BLM HRDC domain consists of five α-helices with a hydrophobic 310-helical loop between helices 1 and 2 and an extended acidic surface comprising residues in helices 3–5. The BLM HRDC domain preferentially binds to ssDNA, though with a markedly low binding affinity (Kd ∼100 μM). NMR chemical shift perturbation studies suggested that the critical DNA-binding residues of the BLM HRDC domain are located in the hydrophobic loop and the N-terminus of helix 2. Interestingly, the isolated BLM HRDC domain had quite different DNA-binding modes between ssDNA and Holliday junctions in electrophoretic mobility shift assay experiments. Based on its surface charge separation and DNA-binding properties, we suggest that the HRDC domain of BLM may be adapted for a unique function among RecQ helicases—that of bridging protein and DNA interactions.

Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain

The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p displays a conserved RNase D core with a flanking HRDC (helicase and RNase D C-terminal) domain in an unusual conformation shown to be important for the processing function of the enzyme. Complexes with AMP and UMP, the products of the RNA degradation process, reveal how the protein specifically recognizes ribonucleotides and their bases. Finally, in vivo mutational studies show the importance of the domain contacts for the processing function of Rrp6p and highlight fundamental differences between the protein and its prokaryotic RNase D counterparts.

Functional analysis of helicase and three tandem HRDC domains of RecQ in Deinococcus radiodurans

RecQ is a highly conserved helicase necessary for maintaining genome stability in all organisms. Genome comparison showed that a homologue of RecQ in Deinococcus radiodurans designated as DR1289 is a member of RecQ family with unusual domain arrangement: a helicase domain, an RecQ C-terminal domain, and surprisingly three HRDC domain repeats, whose function, however, remains obscure currently. Using an insertion deletion, we discovered that the DRRecQ mutation causes an increase in gamma radiation, hydroxyurea and mitomycine C and UV sensitivity. Using the shuttle plasmid pRADK, we complemented various domains of the D. radiodurans RecQ (DRRecQ) to the mutant in vivo. Results suggested that both the helicase and helicase-and-RNase-D-C-terminal (HRDC) domains are essential for complementing several phenotypes. The complementation and biochemical function of DRRecQ variants with different domains truncated in vitro suggested that both the helicase and three HRDC domains are necessary for RecQ functions in D. radiodurans, while three HRDC domains have a synergistic effect on the whole function. Our finding leads to the hypothesis that the RecF recombination pathway is likely a primary path of double strand break repair in this well-known radioresistant organism.

Accumulation of Werner protein at DNA double-strand breaks in human cells

Werner syndrome is an autosomal recessive accelerated-aging disorder caused by a defect in the WRN gene, which encodes a member of the RecQ family of DNA helicases with an exonuclease activity. In vitro experiments have suggested that WRN functions in several DNA repair processes, but the actual functions of WRN in living cells remain unknown. Here, we analyzed the kinetics of the intranuclear mobilization of WRN protein in response to a variety of types of DNA damage produced locally in the nucleus of human cells. A striking accumulation of WRN was observed at laser-induced double-strand breaks, but not at single-strand breaks or oxidative base damage. The accumulation of WRN at double-strand breaks was rapid, persisted for many hours, and occurred in the absence of several known interacting proteins including polymerase beta, poly(ADP-ribose) polymerase 1 (PARP1), Ku80, DNA-dependent protein kinase (DNA-PKcs), NBS1 and histone H2AX. Abolition of helicase activity or deletion of the exonuclease domain had no effect on accumulation, whereas the presence of the HRDC (helicase and RNaseD C-terminal) domain was necessary and sufficient for the accumulation. Our data suggest that WRN functions mainly at DNA double-strand breaks and structures resembling double-strand breaks in living cells, and that an autonomous accumulation through the HRDC domain is the initial response of WRN to the double-strand breaks.

Conferring Substrate Specificity to DNA Helicases: Role of the RecQ HRDC Domain

RecQ DNA helicases are multidomain enzymes that play pivotal roles in genome maintenance pathways. While the ATPase and helicase activities of these enzymes can be attributed to the conserved catalytic core domain, the role of the Helicase-and-RNase-D-C-terminal (HRDC) domain in RecQ function has yet to be elucidated. Here, we report the crystal structure of the E. coli RecQ HRDC domain, revealing a globular fold that resembles known DNA binding domains. We show that this domain preferentially binds single-stranded DNA and identify its DNA binding surface. HRDC domain mutations in full-length RecQ lead to surprising differences in its structure-specific DNA binding properties. These data support a model in which naturally occurring variations in DNA binding residues among diverse RecQ homologs serve to target these enzymes to distinct substrates and provide insight into a mechanism whereby RecQ enzymes have evolved distinct functions in organisms that encode multiple recQ genes.

Crystal Structure of Escherichia coli RNase D, an Exoribonuclease Involved in Structured RNA Processing

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.

The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants.

The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome. Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles. SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ. A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region). In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end. These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe. In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants. In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes. Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes. Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.

Different domains of Sgs1 are required for mitotic and meiotic functions.

The SGS1 of Saccharomyces cerevisiae is a homologue for human Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome causative genes. Disruptants of SGS1 show high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea, and hyper recombination phenotypes including interchromosomal homologous recombination in mitotic growth. In addition, sgs1 disruptants show poor sporulation and a reduced level of meiotic recombination as assayed by return-to-growth. We examined domains of Sgs1 required for mitotic and meiotic functions of Sgs1 by transfecting variously mutated SGS1 into sgs1 disruptants. The N-terminal 1-401 amino acid region was required for complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations of sgs1 disruptants in mitotic growth and for complementation of poor sporulation and of reduced meiotic recombination. Although the N-terminal 1-125 amino acid region was absolutely required for the complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations in mitotic growth, it was dispensable for the meiotic functions. In contrast, the highly acidic region (400-596 amino acid) was dispensable for the mitotic functions but a deletion of this region affected the meiotic functions. The C-terminal 1271-1350 amino acid region containing a HRDC (helicase and RNaseD C-terminal) domain was dispensable for the mitotic and meiotic functions. Although DNA helicase activity of Sgs1 was not required for Sgs1 to complement the meiotic functions, a deletion of helicase motifs III-IV (842-1046 amino acid) abolished the complementing activity of Sgs1, indicating that a structurally intact helicase domain is necessary for Sgs1 to fulfill its meiotic functions.

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

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. 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. 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.