DNA binding by the substrate specificity (wedge) domain of RecG helicase suggests a role in processivity

RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.     

RecG interacts directly with SSB: implications for stalled replication fork regression

RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (−)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression.     

Revealing the DNA Unwinding Activity and Mechanism of Fork Reversal by RecG While Exposed to Variants of Stalled Replication-fork at Single-Molecular Resolution

RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. The elemental mechanism of the helicase activity of RecG with several non-homologous stalled fork structures resembling intermediates formed during the process of DNA repair has been investigated in the present study to capture the dynamic stages of genetic rearrangement. The functional characterization has been exemplified through quantifying the response of the substrate in terms of their molecular heterogeneity and dynamical response by employing single-molecule fluorescence methods. An elevated processivity of RecG is observed for the stalled fork where progression of lagging daughter strand is ahead as compared to that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. RecG is found to adopt an asymmetric mode of locomotion to unwind the lagging daughter strand for facilitating formation of Holliday junction that acts as a suitable intermediate for recombinational repair pathway. Our results emphasize the mechanism adopted by RecG during its ‘sliding back’ mode along the lagging daughter strand to be ‘active translocation and passive unwinding’. This also provide clues as to how this helicase decides and controls the mode of translocation along the DNA to unwind.     

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.     

Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks

In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a 'repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.     

Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.     

Restriction of RecG translocation by DNA mispairing

BackgroundThe RecG DNA helicase plays a crucial role in stalled replication fork rescue. We have recently discovered that interaction of RecG with single-strand DNA binding protein (SSB) remodels RecG, allowing it to spontaneously translocate upstream of the fork. Based on these findings, we hypothesized that mispairing of DNA could limit such translocation of RecG.MethodsHere, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing.ResultsWe found that a CC mispairing, at a distance of 30 bp from the fork position, prevents translocation of RecG over this mispairing. A G-bulge, placed at the same distance, also has a similar blocking efficiency. However, a CC mispairing, 10 bp away from the fork, does not prevent RecG translocation beyond 10 bp distance, but decreases complex yield. Modeling of RecG-DNA complexes show that 10 bp distance from the fork is within the binding footprint of RecG on DNA.ConclusionsOur results suggest that the RecG translocation upstream of the replication fork is limited by mispairings in the parental arm of the replication fork.General significanceThese findings led us to propose dual functions for RecG, in which the thermally driven translocation of RecG can be a mechanism for the additional control of the DNA paring in which RecG can detect the lesions in front of the replication fork, adding to the fidelity of the DNA replication machinery.     

Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.     

Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

When replication stalls and forks disassemble, the restart primosome is required to reload the replicative helicase so that chromosomal replication can be reinitiated. We have taken a photo-cross-linking approach, using model replication forks containing a phenyl diazirine placed at single locations, to determine the positions of primosomal protein binding and changes in interactions that occur during the assembly reaction. This approach revealed a novel mode for single-stranded DNA-binding protein (SSB)-DNA binding, in which SSB interacts with both the leading and lagging single-strand segments and the parental duplex of the fork. Cross-linking to a novel region within SSB is observed only when it is bound to forked structures. This binding mode is also followed by PriB. PriA binds to the fork, excluding SSB and PriB, interacting with the primer terminus, single-stranded leading and lagging strands and duplex in immediate proximity of the fork. SSB binds to flanking single-stranded segments distal to the fork in the presence of PriA. The addition of PriB or DnaT to a PriA-SSB-fork complex does not lead to cross-linking or displacement, suggesting that their association is through protein-protein interactions at early stages of the reaction. Upon addition of DnaC and the DnaB helicase in the presence of ATPγS, helicase is assembled, leading to contacts within the duplex region on the tracking (lagging) strand and strong contacts with the displaced leading single strand near the fork. PriA is displaced from DNA upon helicase assembly.     

Stabilization of a stalled replication fork by concerted actions of two helicases

PriA helicase plays crucial roles in restoration of arrested replication forks. It carries a "3' terminus binding pocket" in its N-terminal DNA binding domain, which is required for high affinity binding of PriA to a fork carrying a 3'-end of a nascent leading strand at the branch. We show that the abrogation of the 3' terminus recognition either by a mutation in the 3' terminus binding pocket or by the bulky modification of the 3'-end leads to unwinding of the unreplicated duplex arm on this fork, causing potential fork destabilization. This indicates a critical role of the 3' terminus binding pocket of PriA in its "stable" binding at the fork for primosome assembly. In contrast, PriA unwinds the unreplicated duplex region on a fork without a 3'-end, potentially destabilizing the fork. However, this process is inhibited by RecG helicase, capable of regressing the fork until the 3'-end of the nascent leading strand reaches the branch. PriA now stably binds to this regressed fork, stabilizing it. Using a model arrest-fork-substrate, we reconstitute the above process in vitro with RecG and PriA proteins. Our results present a novel mechanism by which two helicases function in a highly coordinated manner to generate a structure in which an arrested fork is stabilized for further repair and/or replication restart.     

枯草芽孢杆菌全长引物酶的溶液结构研究

在细菌DNA复制中,DnaG引物酶合成RNA引物,然后合成的引物通过DNA聚合酶进行延伸. DnaG引物酶由3个结构域组成,N端锌结合结构域（zinc-binding domain,ZBD）、RNA聚合酶结构域（RNA polymerase domain,RPD）和C端解旋酶结合结构域（helicase binding domain,HBD）. 在合成引物的过程中,引物酶的3个结构域协同作用,缺一不可. 尽管引物酶3个结构域的结构均已有研究报道,但到目前为止,引物酶的全长结构尚不清楚. 我们在上海光源利用小角X射线散射技术研究了枯草芽孢杆菌全长引物酶的溶液结构,首次构建了全长引物酶结构模型. 我们发现,枯草芽孢杆菌引物酶在溶液中处于伸展状态,且ZBD和HBD结构域相对于RPD结构域呈现出连续的构象变化. 本文研究表明DnaG引物酶中的结构域重排可能有助于其在DNA复制中发挥功能.     

Fork regression is an active helicase‐driven pathway in bacteriophage T4

Reactivation of stalled replication forks requires specialized mechanisms that can recognize the fork structure and promote downstream processing events. Fork regression has been implicated in several models of fork reactivation as a crucial processing step that supports repair. However, it has also been suggested that regressed forks represent pathological structures rather than physiological intermediates of repair. To investigate the biological role of fork regression in bacteriophage T4, we tested several mechanistic models of regression: strand exchange‐mediated extrusion, topology‐driven fork reversal and helicase‐mediated extrusion. Here, we report that UvsW, a T4 branch‐specific helicase, is necessary for the accumulation of regressed forks in vivo, and that UvsW‐catalysed regression is the dominant mechanism of origin‐fork processing that contributes to double‐strand end formation. We also show that UvsW resolves purified fork intermediates in vitro by fork regression. Regression is therefore part of an active, UvsW‐driven pathway of fork processing in bacteriophage T4.     

Stalled replication forks: Making ends meet for recognition and stabilization

In bacteria, PriA protein, a conserved DEXH‐type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA‐binding property allows it to recognize and stabilize stalled forks and the structures derived from them. Cells must cope with fork stalls caused by various replication stresses to complete replication of the entire genome. Failure of the stalled fork stabilization process and eventual restart could lead to various forms of genomic instability. The low viability of priA null cells indicates a frequent occurrence of fork stall during normal growth that needs to be properly processed. PriA specifically recognizes the 3′‐terminus of the nascent leading strand or the invading strand in a displacement (D)‐loop by the three‐prime terminus binding pocket (TT‐pocket) present in its unique DNA binding domain. Elucidation of the structural basis for recognition of arrested forks by PriA should provide useful insight into how stalled forks are recognized in eukaryotes.     

Electron microscopy of DNA.helicase-I complexes in the act of strand separation

Electron microscopy was used to characterize the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase I. Linear DNA with 5'-protruding strands as well as single-stranded gaps was incubated, under unwinding assay conditions, with the helicase. E. coli single-stranded-DNA-binding protein (SSB) was added to order the denatured DNA. Up to 70% of the sites of SSB-complexed DNA were observed as forks. The position of the strand-separating enzyme was indicated by a gap in the complex between fork and SSB on that arm which initially provided the binding site. The complex between DNA and helicase varied in length although in all cases it was long enough to comprise several helicase I molecules. A mutant helicase I (helicase I del29) which, unlike the wild-type enzyme, fails to show cooperative DNA-binding behaviour was found to prevent an abnormally short stretch of DNA near the fork from binding SSB. Apparently, one or very few helicase molecules would be sufficient for the opening of a DNA duplex although, typically, the fork is shifted by a tract of helicase I molecules. SSB displaces helicase I from single-stranded DNA but fails to do so from a fork or a single-strand/double-strand junction. The difference is consistent with the observation that SSB does not inhibit the unwinding reaction despite its rapid association with the separated strands. Helicase I unwinds in the 5'-3' direction of the bound strand. Observations so far indicate that the enzyme exploits the single strand at the initial DNA-binding site for orienting its action, and not the complementary, completely base-paired strand.     

The intrinsically disordered linker of E. coli SSB is critical for the release from single‐stranded DNA

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single‐stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB‐interactome. Key to both roles is the C‐terminal, one‐third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB–SSB interactions, and when the IDL is removed a terminal SSB–DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.     

RecQ and RecG helicases have distinct roles in maintaining the stability of polypurine.polypyrimidine sequences

DNA triplex structures can block the replication fork and result in double-stranded DNA breaks (DSBs). RecQ and RecG helicases may be important for replication of such sequences as RecQ resolves synthetic triplex DNA structures and RecG mediates replication restart by fork regression. Primer extension on an 88bp triplex-forming polypurine.polypyrimidine (Pu.Py) tract from the PKD1 gene demonstrated that RecQ, but not RecG, facilitated primer extension by T7 DNA polymerase. A high-throughput, dual plasmid screening system using isogenic bacterial lines deficient in RecG, RecQ, or both, revealed that RecQ deficiency increased mutation to sequence flanking this 88bp tract by eight to ten-fold. Although RecG facilitated small deletions in an 88bp mirror repeat-containing sequence, it was absolutely required to maintain a 2.5kb Pu.Py tract containing multiple mirror repeats. These results support a two-tiered model where RecQ facilitates fork progression through triplex-forming tracts and, failing processivity, RecG is critical for replication fork restart.     

Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein with increased processivity

In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins at the replication fork. Single-stranded DNA binding protein (SSB) is an essential protein that binds tightly and cooperatively to single-stranded DNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase with its cognate SSB via a short six amino acid linker increases affinity for primer-template DNA by sixfold and subsequently increases processivity by sevenfold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.     

Novel, fluorescent, SSB protein chimeras with broad utility

The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use.