Biochemical properties of naturally occurring human bloom helicase variants.

Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core (BLMcore, residues 636-1298) with either the P868L or G1120R substitution. We also purified a BLMcore K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLMcore P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLMcore P868L and G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLMcore K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLMcore. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities.

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD-SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. We found that RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD:SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro. Additionally, mutant Escherichia coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG, although the phenotypes of the SSB-binding radD mutants are not as severe as a full radD deletion. This suggests that cellular RadD requires an intact interaction with SSB for full RadD function.

Biochemical Properties of Naturally Occurring Human Bloom Helicase Variants.

Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo , there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro , we purified the BLM catalytic core (BLM core , residues 636-1298) with either the P868L or G1120R substitution. We also purified a BLM core K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLM core P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLM core P868L and G1120R retain helicase function in vitro , it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo . Interestingly, we found that BLM core K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLM core . Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.

X-ray crystal structure of the Escherichia coli RadD DNA repair protein bound to ADP reveals a novel zinc ribbon domain.

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zinc-ribbon element that was not modelled in a previous RadD crystal structure. Insights into the mode of nucleotide binding and additional structure refinement afforded by the new RadD model will help to drive investigations into the activity of RadD as a genome stability and repair factor.

Natural Transformation Protein ComFA Exhibits Single-Stranded DNA Translocase Activity.

Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a few proteins required for natural transformation in Gram-positive bacteria. Its structural resemblance to the DEAD box helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5' to 3'. However, this translocase activity does not lead to DNA unwinding under the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake. IMPORTANCE Competence, or the ability of bacteria to take up and incorporate foreign DNA in a process called natural transformation, is common in the bacterial kingdom. Research in several bacterial species suggests that long, contiguous stretches of DNA are imported into cells in a processive manner, but how bacteria power transformation remains unclear. Our finding that ComFA, a DEAD box helicase required for competence in Gram-positive bacteria, translocates on single-stranded DNA from 5' to 3', supports the long-held hypothesis that ComFA may be the motor powering DNA transport during natural transformation. Moreover, ComFA may be a previously unidentified type of DEAD box helicase-one with the capability of extended translocation on single-stranded DNA.

The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader.

In many bacteria and eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+(ATPases Associated with various cellular Activities) ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of Escherichia coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We have identified a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate that elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as in polymerase clamp loading and certain classes of DNA transposases.

E. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands.

G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.

An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase.

Helicases couple ATP hydrolysis to nucleic acid binding and unwinding via molecular mechanisms that remain poorly defined for most enzyme subfamilies within the superfamily 2 (SF2) helicase group. A crystal structure of the PriA SF2 DNA helicase, which governs restart of prematurely terminated replication processes in bacteria, revealed the presence of an aromatic-rich loop (ARL) on the presumptive DNA-binding surface of the enzyme. The position and sequence of the ARL was similar to loops known to couple ATP hydrolysis with DNA binding in a subset of other SF2 enzymes, however, the roles of the ARL in PriA had not been investigated. Here, we show that changes within the ARL sequence uncouple PriA ATPase activity from DNA binding. In vitro protein-DNA crosslinking experiments define a residue- and nucleotide-specific interaction map for PriA, showing that the ARL binds replication fork junctions whereas other sites bind the leading or lagging strands. We propose that DNA binding to the ARL allosterically triggers ATP hydrolysis in PriA. Additional SF2 helicases with similarly positioned loops may also couple DNA binding to ATP hydrolysis using related mechanisms.

A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.

Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases.

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

The BLM dissolvasome in DNA replication and repair.

RecQ DNA helicases are critical for proper maintenance of genomic stability, and mutations in multiple human RecQ genes are linked with genetic disorders characterized by a predisposition to cancer. RecQ proteins are conserved from prokaryotes to humans and in all cases form higher-order complexes with other proteins to efficiently execute their cellular functions. The focus of this review is a conserved complex that is formed between RecQ helicases and type-I topoisomerases. In humans, this complex is referred to as the BLM dissolvasome or BTR complex, and is comprised of the RecQ helicase BLM, topoisomerase IIIα, and the RMI proteins. The BLM dissolvasome functions to resolve linked DNA intermediates without exchange of genetic material, which is critical in somatic cells. We will review the history of this complex and highlight its roles in DNA replication, recombination, and repair. Additionally, we will review recently established interactions between BLM dissolvasome and a second set of genome maintenance factors (the Fanconi anemia proteins) that appear to allow coordinated genome maintenance efforts between the two systems.

Structure and biochemical activities of Escherichia coli MgsA.

Bacterial "maintenance of genome stability protein A" (MgsA) and related eukaryotic enzymes play important roles in cellular responses to stalled DNA replication processes. Sequence information identifies MgsA enzymes as members of the clamp loader clade of AAA+ proteins, but structural information defining the family has been limited. Here, the x-ray crystal structure of Escherichia coli MgsA is described, revealing a homotetrameric arrangement for the protein that distinguishes it from other clamp loader clade AAA+ proteins. Each MgsA protomer is composed of three elements as follows: ATP-binding and helical lid domains (conserved among AAA+ proteins) and a tetramerization domain. Although the tetramerization domains bury the greatest amount of surface area in the MgsA oligomer, each of the domains participates in oligomerization to form a highly intertwined quaternary structure. Phosphate is bound at each AAA+ ATP-binding site, but the active sites do not appear to be in a catalytically competent conformation due to displacement of Arg finger residues. E. coli MgsA is also shown to form a complex with the single-stranded DNA-binding protein through co-purification and biochemical studies. MgsA DNA-dependent ATPase activity is inhibited by single-stranded DNA-binding protein. Together, these structural and biochemical observations provide insights into the mechanisms of MgsA family AAA+ proteins.

Peptide inhibitors identify roles for SSB C-terminal residues in SSB/exonuclease I complex formation.

Bacterial single-stranded (ss) DNA-binding proteins (SSBs) facilitate DNA replication, recombination, and repair processes in part by recruiting diverse genome maintenance enzymes to ssDNA. This function utilizes the C-terminus of SSB (SSB-Ct) as a common binding site for SSB's protein partners. The SSB-Ct is a highly conserved, amphipathic sequence comprising acidic and hydrophobic elements. A crystal structure of Escherichia coli exonuclease I (ExoI) bound to a peptide comprising the E. coli SSB-Ct sequence shows that the C-terminal-most SSB-Ct Phe anchors the peptide to a binding pocket on ExoI and implicates electrostatic binding roles for the acidic SSB-Ct residues. Here, we use SSB-Ct peptide variants in competition experiments to examine the roles of individual SSB-Ct residues in binding ExoI in solution. Altering the C-terminal-most Pro or Phe residues in the SSB-Ct strongly impairs SSB-Ct binding to ExoI, confirming a major role for the hydrophobic SSB-Ct residues in binding ExoI. Alteration of N-terminal SSB-Ct residues leads to changes that reflect cumulative electrostatic binding roles for the Asp residues in SSB-Ct. The SSB-Ct peptides also abrogate SSB stimulation of ExoI activity through a competitive inhibition mechanism, indicating that the peptides can disrupt ExoI/SSB/ssDNA ternary complexes. Differences in the potency of the SSB-Ct peptide variants in the binding and nuclease inhibition studies indicate that the acidic SSB-Ct residues play a more prominent role in the context of the ternary complex than in the minimal ExoI/SSB-Ct interaction. Together, these data identify roles for residues in the SSB-Ct that are important for SSB complex formation with its protein partners.

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.

Sit down, relax and unwind: structural insights into RecQ helicase mechanisms.

Helicases are specialized molecular motors that separate duplex nucleic acids into single strands. The RecQ family of helicases functions at the interface of DNA replication, recombination and repair in bacterial and eukaryotic cells. They are key, multifunctional enzymes that have been linked to three human diseases: Bloom's, Werner's and Rothmund-Thomson's syndromes. This review summarizes recent studies that relate the structures of RecQ proteins to their biochemical activities.

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.

Coupling DNA-binding and ATP hydrolysis in Escherichia coli RecQ: role of a highly conserved aromatic-rich sequence.

RecQ enzymes are broadly conserved Superfamily-2 (SF-2) DNA helicases that play critical roles in DNA metabolism. RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA-binding/unwinding are unknown. In many Superfamily-1 (SF-1) DNA helicases, helicase sequence motif III links these activities by binding both single-stranded (ss) DNA and ATP. However, the ssDNA-binding aromatic-rich element in motif III present in these enzymes is missing from SF-2 helicases, raising the question of how these enzymes link ATP hydrolysis to DNA-binding/unwinding. We show that Escherichia coli RecQ contains a conserved aromatic-rich loop in its helicase domain between motifs II and III. Although placement of the RecQ aromatic-rich loop is topologically distinct relative to the SF-1 enzymes, both loops map to similar tertiary structural positions. We examined the functions of the E.coli RecQ aromatic-rich loop using RecQ variants with single amino acid substitutions within the segment. Our results indicate that the aromatic-rich loop in RecQ is critical for coupling ATPase and DNA-binding/unwinding activities. Our studies also suggest that RecQ's aromatic-rich loop might couple ATP hydrolysis to DNA-binding in a mechanistically distinct manner from SF-1 helicases.

PriB stimulates PriA helicase via an interaction with single-stranded DNA.

The frequency with which replication forks break down in all organisms requires that specific mechanisms ensure completion of genome duplication. In Escherichia coli a major pathway for reloading of the replicative apparatus at sites of fork breakdown is dependent on PriA helicase. PriA acts in conjunction with PriB and DnaT to effect loading of the replicative helicase DnaB back onto the lagging strand template, either at stalled fork structures or at recombination intermediates. Here we showed that PriB stimulates PriA helicase, acting to increase the apparent processivity of PriA. This stimulation correlates with the ability of PriB to form a ternary complex with PriA and DNA structures containing single-stranded DNA, suggesting that the known single-stranded DNA binding function of PriB facilitates unwinding by PriA helicase. This enhanced apparent processivity of PriA might play an important role in generating single-stranded DNA at stalled replication forks upon which to load DnaB. However, stimulation of PriA by PriB is not DNA structure-specific, demonstrating that targeting of stalled forks and recombination intermediates during replication restart likely resides with PriA alone.

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.