HRDC Domain Research Articles

UvrD2 Is Essential in Mycobacterium tuberculosis, but Its Helicase Activity Is Not Required

UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosis has two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2 gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2 is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2 at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.

Activities of human RRP6 and structure of the human RRP6 catalytic domain

The eukaryotic RNA exosome is a highly conserved multi-subunit complex that catalyzes degradation and processing of coding and noncoding RNA. A noncatalytic nine-subunit exosome core interacts with Rrp44 and Rrp6, two subunits that possess processive and distributive 3'-to-5' exoribonuclease activity, respectively. While both Rrp6 and Rrp44 are responsible for RNA processing in budding yeast, Rrp6 may play a more prominent role in processing, as it has been demonstrated to be inhibited by stable RNA secondary structure in vitro and because the null allele in budding yeast leads to the buildup of specific structured RNA substrates. Human RRP6, otherwise known as PM/SCL-100 or EXOSC10, shares sequence similarity to budding yeast Rrp6 and is proposed to catalyze 3'-to-5' exoribonuclease activity on a variety of nuclear transcripts including ribosomal RNA subunits, RNA that has been poly-adenylated by TRAMP, as well as other nuclear RNA transcripts destined for processing and/or destruction. To characterize human RRP6, we expressed the full-length enzyme as well as truncation mutants that retain catalytic activity, compared their activities to analogous constructs for Saccharomyces cerevisiae Rrp6, and determined the X-ray structure of a human construct containing the exoribonuclease and HRDC domains that retains catalytic activity. Structural data show that the human active site is more exposed when compared to the yeast structure, and biochemical data suggest that this feature may play a role in the ability of human RRP6 to productively engage and degrade structured RNA substrates more effectively than the analogous budding yeast enzyme.

Pleiotropic effects of RecQ in Deinococcus radiodurans

In Deinococcus radiodurans, there is a unique RecQ homolog (DR1289) with three-tandem HRDC domains. Deletion of drrecQ resulted in a low doubling rate and sensitivity to hydrogen peroxide. Here, we used cDNA microarray and biochemical assays to explore the physiological changes in the drrecQ mutant. The expressions of genes with predicted functions involved in iron homeostasis, antioxidant system, electron transport, and energy metabolism were significantly altered in response to drrecQ disruption. More reactive oxygen species (ROS) was accumulated in drrecQ mutant strain when compared to wild type. In addition, ICP-MS results showed that the intracellular level of iron was relatively higher, whereas the concentration of manganese was lower in drrecQ mutant than in wild type. Furthermore, our microarray data and pulsed-field gel results showed that DNA suffered more damage in drrecQ mutant than in wild type under 20 mM hydrogen peroxide stress. These results suggested that drrecQ is a gene of pleiotropic functions and contributes to the extraordinary resistance of D. radiodurans against stresses.

RecQ DNA helicase HRDC domains are critical determinants in Neisseria gonorrhoeae pilin antigenic variation and DNA repair

Neisseria gonorrhoeae (Gc), an obligate human bacterial pathogen, utilizes pilin antigenic variation to evade host immune defences. Antigenic variation is driven by recombination between expressed (pilE) and silent (pilS) copies of the pilin gene, which encodes the major structural component of the type IV pilus. We have investigated the role of the GcRecQ DNA helicase (GcRecQ) in this process. Whereas the vast majority of bacterial RecQ proteins encode a single 'Helicase and RNase D C-terminal' (HRDC) domain, GcRecQ encodes three tandem HRDC domains at its C-terminus. Gc mutants encoding versions of GcRecQ with either two or all three C-terminal HRDC domains removed are deficient in pilin variation and sensitized to UV light-induced DNA damage. Biochemical analysis of a GcRecQ protein variant lacking two HRDC domains, GcRecQDeltaHRDC2,3, shows it has decreased affinity for single-stranded and partial-duplex DNA and reduced unwinding activity on a synthetic Holliday junction substrate relative to full-length GcRecQ in the presence of Gc single-stranded DNA-binding protein (GcSSB). Our results demonstrate that the multiple HRDC domain architecture in GcRecQ is critical for structure-specific DNA binding and unwinding, and suggest that these features are central to GcRecQ's roles in Gc antigenic variation and DNA repair.

Intrinsic ssDNA Annealing Activity in the C-Terminal Region of WRN

Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3' to 5' exonuclease, 3' to 5' helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072-1150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.

Crystal structure of the HRDC domain of human Werner syndrome protein, WRN

Crystal structure of the HRDC domain of human Werner syndrome protein, WRN

Domain Requirements for DNA Unwinding by Mycobacterial UvrD2, an Essential DNA Helicase

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.

Structure and function of the regulatory C-terminal HRDC domain from Deinococcus radiodurans RecQ

RecQ helicases are critical for maintaining genome integrity in organisms ranging from bacteria to humans by participating in a complex network of DNA metabolic pathways. Their diverse cellular functions require specialization and coordination of multiple protein domains that integrate catalytic functions with DNA–protein and protein–protein interactions. The RecQ helicase from Deinococcus radiodurans (DrRecQ) is unusual among RecQ family members in that it has evolved to utilize three ‘Helicase and RNaseD C-terminal’ (HRDC) domains to regulate its activity. In this report, we describe the high-resolution structure of the C-terminal-most HRDC domain of DrRecQ. The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains. Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate. Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.

Three tandem HRDC domains have synergistic effect on the RecQ functions in Deinococcus radiodurans

The RecQ family of DNA helicases performs essential functions in the maintenance of genomic stability in all organisms. In Deinococcus radiodurans, DR1289 is a special member of RecQ family with unique arrangement of three tandem HRDC domains in the C-terminus. A dr1289 mutant is hypersensitive to γ-irradiation, UV, H 2O 2 and mitomycin C. By complementing the dr1289 mutant with various domains of Dr1289 in vivo, we have determined that the helicase and all three HRDC domains are indispensable for complete DNA damage resistance. Using a continuous fluorescent dye-displacement assay, we investigated the optimal conditions for Dr1289 unwinding function at various concentrations of ATP and metal ions to show that the helicase activity is comparable to what observed in Escherichia coli RecQ. We also found that the helicase domain is necessary for the unwinding and ATPase activity and that the three tandem HRDC domains increase the efficiency of these activities. Based on these data, we propose that the C-terminus of Dr1289 has evolved in D. radiodurans to confront the types and amounts of DNA damage.

The Bloom's Syndrome Helicase Can Promote the Regression of a Model Replication Fork

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called "chicken foot" structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges.

BLM is an early responder to DNA double-strand breaks

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a marked predisposition to cancer and elevated genomic instability. The defective protein in BS, BLM, is a member of the RecQ helicase family and is believed to function in various DNA transactions, including in replication, repair, and recombination. Here, we show that both endogenous and overexpressed human BLM accumulates at sites of laser light-induced DNA double-strand breaks within 10 s and colocalizes with γH2AX and ATM. Like its RecQ helicase family member, WRN, the defective protein in Werner syndrome, dissection of the BLM protein revealed that its HRDC domain is sufficient for its recruitment to the damaged sites. In addition, we confirmed that the C-terminal region spanning amino acids 1250–1292 within the HRDC domain is necessary for BLM recruitment. To identify additional proteins required for the recruitment of BLM, we examined the recruitment of BLM in various mutants generated from chicken DT40 cells and found that the early accumulation of BLM was not dependent on the presence of ATM, RAD17, DNA-PKcs, NBS1, XRCC3, RAD52, RAD54, or WRN. Thus, HRDC domain in DNA helicases is a common early responder to DNA double-strand breaks, enabling BLM and WRN to be involved in DNA repair.

Structural Biology of RNA Polymerase III: Subcomplex C17/25 X-Ray Structure and 11 Subunit Enzyme Model

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.

Three HRDC Domains Differentially Modulate Deinococcus radiodurans RecQ DNA Helicase Biochemical Activity

RecQ helicases are key genome maintenance enzymes that function in DNA replication, recombination, and repair. In contrast to nearly every other identified RecQ family member, the RecQ helicase from the radioresistant bacterium Deinococcus radiodurans encodes three "Helicase and RNase D C-terminal" (HRDC) domains at its C terminus. HRDC domains have been implicated in structure-specific nucleic acid binding with roles in targeting RecQ proteins to particular DNA structures; however, only RecQ proteins with single HRDC domains have been examined to date. We demonstrate that the HRDC domains can be proteolytically removed from the D. radiodurans RecQ (DrRecQ) C terminus, consistent with each forming a structural domain. Using this observation as a guide, we produced a panel of recombinant DrRecQ variants lacking combinations of its HRDC domains to investigate their biochemical functions. The N-terminal-most HRDC domain is shown to be critical for high affinity DNA binding and for efficient unwinding of DNA in some contexts. In contrast, the more C-terminal HRDC domains attenuate the DNA binding affinity and DNA-dependent ATP hydrolysis rate of the enzyme and play more complex roles in structure-specific DNA unwinding. Our results indicate that the multiple DrRecQ HRDC domains have evolved to encode DNA binding and regulatory functions in the enzyme.

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.

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.

Structural and functional homology between the RNAP(I) subunits A14/A43 and the archaeal RNAP subunits E/F.

In the archaeal RNA polymerase and the eukaryotic RNA polymerase II, two subunits (E/F and RPB4/RPB7, respectively) form a heterodimer that reversibly associates with the core of the enzyme. Recently it has emerged that this heterodimer also has a counterpart in the other eukaryotic RNA polymerases: in particular two subunits of RNA polymerase I (A14 and A43) display genetic and biochemical characteristics that are similar to those of the RPB4 and RPB7 subunits, despite the fact that only A43 shows some sequence homology to RPB7. We demonstrate that the sequence of A14 strongly suggests the presence of a HRDC domain, a motif that is found at the C-terminus of a number of helicases and RNases. The same motif is also seen in the structure of the F subunit, suggesting a structural link between A14 and the RPB4/C17/subunit F family, even in the absence of direct sequence homology. We show that it is possible to co-express and co-purify large amounts of the recombinant A14/A43 heterodimer, indicating a tight and specific interaction between the two subunits. To shed light on the function of the heterodimer, we performed gel mobility shift assays and showed that the A14/A43 heterodimer binds single-stranded RNA in a similar way to the archaeal E/F complex.

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 ATPγS. 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 λ 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.

ARIA: automated NOE assignment and NMR structure calculation.

In the light of several ongoing structural genomics projects, faster and more reliable methods for structure calculation from NMR data are in great demand. The major bottleneck in the determination of solution NMR structures is the assignment of NOE peaks (nuclear Overhauser effect). Due to the high complexity of the assignment problem, most NOEs cannot be directly converted into unambiguous inter-proton distance restraints. We present version 1.2 of our program ARIA (Ambiguous Restraints for Iterative Assignment) for automated assignment of NOE data and NMR structure calculation. We summarize recent progress in correcting for spin diffusion with a relaxation matrix approach, representing non-bonded interactions in the force field and refining final structures in explicit solvent. We also discuss book-keeping, data exchange with spectra assignment programs and deposition of the analysed experimental data to the databases. ARIA 1.2 is available from: http://www.pasteur.fr/recherche/unites/Binfs/aria/. XML DTDs (for chemical shifts and NOE crosspeaks), Python scripts for the conversion of various NMR data formats and the results of example calculations using data from the S. cerevisiae HRDC domain are available from: http://www.pasteur.fr/recherche/unites/Binfs/aria/

Refinement of the protein backbone angle psi in NMR structure calculations.

Cross-correlated relaxation rates involving the Calpha-Halpha dipolar interaction and the carbonyl (C') chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle psi can be directly refined against such cross-correlated relaxation rates (gammaHalphaCalpha,C') and the three-bond H/D isotope effect on the Calpha chemical shifts (3 deltaCalpha(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle psi is defined. We have applied the new refinement method to the alpha-Spectrin SH3 domain (a beta-sheet protein) and to the Sgs1p HRDC domain (an alpha-helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the psi-refined NMR structures of the SH3 domain deviate less from the 1.8 A crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.

A novel NMR experiment for the sequential assignment of proline residues and proline stretches in 13C/15N-labeled proteins.

A new pulse sequence is described for the sequential assignment of proline residues in 13C/15N-labeled proteins by correlating C delta and C alpha chemical shifts of proline residues with the H alpha chemical shift of the preceding residue. Notably, the experiment can provide the sequential connectivities in poly-proline stretches, which cannot be determined using standard triple resonance experiments. Excellent solvent suppression is achieved by coherence selection via a heteronuclear gradient echo. The new pulse sequence has been successfully applied to the 11 kDa HRDC domain.