Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.     

RuvA is a sliding collar that protects Holliday junctions from unwinding while promoting branch migration

The RuvAB proteins catalyze branch migration of Holliday junctions during DNA recombination in Escherichia coli. RuvA binds tightly to the Holliday junction, and then recruits two RuvB pumps to power branch migration. Previous investigations have studied RuvA in conjunction with its cellular partner RuvB. The replication fork helicase DnaB catalyzes branch migration like RuvB but, unlike RuvB, is not dependent on RuvA for activity. In this study, we specifically analyze the function of RuvA by studying RuvA in conjunction with DnaB, a DNA pump that does not work with RuvA in the cell. Thus, we use DnaB as a tool to dissect RuvA function from RuvB. We find that RuvA does not inhibit DnaB-catalyzed branch migration of a homologous junction, even at high concentrations of RuvA. Hence, specific protein-protein interaction is not required for RuvA mobilization during branch migration, in contrast to previous proposals. However, low concentrations of RuvA block DnaB unwinding at a Holliday junction. RuvA even blocks DnaB-catalyzed unwinding when two DnaB rings are acting in concert on opposite sides of the junction. These findings indicate that RuvA is intrinsically mobile at a Holliday junction when the DNA is undergoing branch migration, but RuvA is immobile at the same junction during DNA unwinding. We present evidence that suggests that RuvA can slide along a Holliday junction structure during DnaB-catalyzed branch migration, but not during unwinding. Thus, RuvA may act as a sliding collar at Holliday junctions, promoting DNA branch migration activity while blocking other DNA remodeling activities. Finally, we show that RuvA is less mobile at a heterologous junction compared to a homologous junction, as two opposing DnaB pumps are required to mobilize RuvA over heterologous DNA.     

Polarity and bypass of DNA heterology during branch migration of Holliday junctions by human RAD54, BLM, and RECQ1 proteins

Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3'→5' direction, whereas RuvAB drives it in the 5'→3' direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.     

Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli.

Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks. Two key steps in this process are the branch migration of Holliday junctions followed by their resolution into mature recombination products. In E.coli, branch migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease. Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes. Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity. Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed branch migration. The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism.     

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.     

Simultaneous binding to the tracking strand,displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase

Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding.     

A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase

Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.     

The phage T4 protein UvsW drives Holliday junction branch migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.     

A pivotal role for the structure of the Holliday junction in DNA branch migration.

Branch migration of a DNA Holliday junction is a key step in genetic recombination that affects the extent of transfer of genetic information between homologous DNA sequences. We previously observed that the rate of spontaneous branch migration is exceedingly sensitive to metal ions and postulated that the structure of the cross-over point might be one critical determinant of the rate of branch migration. Other investigators have shown that in the presence of divalent metal ions like magnesium, the Holliday junction assumes a folded conformation in which base stacking is retained through the cross-over point. This base stacking is disrupted in the absence of magnesium. Here we measure the rate of branch migration as a function of Mg2+ concentration. The rate of branch migration increases dramatically at MgCl2 concentrations below 500 microM, with the steepest acceleration occurring between 300 and 100 microM MgCl2. This increase in the rate of branch migration coincides with the loss of base stacking in the four-way junction over this same interval of magnesium concentration, as measured by the susceptibility of junction residues to modification by osmium tetroxide and diethyl pyrocarbonate. We conclude that at physiological concentrations of intracellular Mg2+, base stacking in the Holliday junction constitutes one kinetic barrier to branch migration and that disruption of base stacking at the cross-over relieves this constraint.     

Functional analysis of DNA replication fork reversal catalyzed by Mycobacterium tuberculosis RuvAB proteins

Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins.     

Energy flow considerations and thermal fluctuational opening of DNA base pairs at a replicating fork: unwinding consistent with observed replication rates.

The effect of an open loop of various sizes on the thermal stability of the adjoining intact base pairs in a duplex DNA chain is studied in a lattice model of Poly(dG).Poly(dC). We find that for a Y-shaped fork configuration the thermal fluctuation at the fork is so enhanced that the life time of the adjoining base pair is much smaller than the 1 millisecond time scale associated with helicase separation of a base pair in some systems. Our analysis indicates that thermal fluctuational base pair opening may be of importance in facilitating the enzyme unwinding process during chain elongation of a replicating DNA. It is most likely that the thermal fluctuational opening of the base pair at the junction of a replicating fork is fast enough so that a DNA unwinding enzyme can encounter an unstacked base pair with reasonable probability. This conclusion can explain several experimental observations regarding the temporal relationship between ATP hydrolysis by accessory proteins and primer elongation by a holoenzyme complex in ssDNA. We also discuss a mechanism by which the energy associated with ATP hydrolysis may enhance the thermal driven base opening mechanism.     

DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands

DnaB is a ring-shaped, hexameric helicase that unwinds the E. coli DNA replication fork while encircling one DNA strand. This report demonstrates that DnaB can also encircle both DNA strands and then actively translocate along the duplex. With two strands positioned inside its central channel, DnaB translocates with sufficient force to displace proteins tightly bound to DNA with no resultant DNA unwinding. Thus, DnaB may clear proteins from chromosomal DNA. Furthermore, while encircling two DNA strands, DnaB can drive branch migration of a synthetic Holliday junction with heterologous duplex arms, suggesting that DnaB may be directly involved in DNA recombination in vivo. DnaB binds to just one DNA strand during branch migration. T7 phage gp4 protein also drives DNA branch migration, suggesting this activity generalizes to other ring-shaped helicases.     

Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase

We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.     

RNA structural rearrangement via unwinding and annealing by the cyanobacterial RNA helicase, CrhR

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.     

Bound Lac repressor protein differentially inhibits the unwinding reactions catalyzed by DNA helicases.

A partial duplex DNA substrate containing the Lac repressor binding site, within the duplex region, was constructed to examine the effect of bound Lac repressor on the unwinding reaction catalyzed by several DNA helicases. The substrate contained 90 base pairs of double-stranded DNA and, in the absence of Lac repressor, was effectively unwound by each of the seven helicases tested. The unwinding reactions catalyzed by Escherichia coli Rep protein, bacteriophage T4 Dda protein and E. coli DNA helicase I were not inhibited by the presence of bound Lac repressor. Both SV40 T antigen and E. coli helicase II were partially inhibited by bound repressor at the highest repressor concentrations tested. The helicase reactions catalyzed by E. coli DnaB protein and helicase IV were substantially inhibited by the presence of bound protein. When the length of the duplex region was increased to 323 base pairs the inhibition spectrum caused by bound Lac repressor on the unwinding reactions catalyzed by DnaB protein, helicase I and helicase II was essentially the same as that observed using the shorter partial duplex molecule. Inhibition of the unwinding reaction was due to the presence of bound Lac repressor as evidenced by the substantially weaker inhibition of helicase IV by Lac repressor in the presence of IPTG. In addition, we have shown that Rep protein displaces the bound repressor protein during the course of an unwinding reaction.     

Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements

The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity. The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates. The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C. For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction. The minimum length of the single-stranded tail was determined to be less than 5 nucleotides. Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction. This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase. Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome. The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.     

Nucleic acid unwinding by hepatitis C virus and bacteriophage t7 helicases is sensitive to base pair stability

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.     

Energy Flow Considerations and Thermal Fluctuational Opening of DNA Base Pairs at a Replicating Fork: Unwinding Consistent with Observed Replication Rates

Abstract

The effect of an open loop of various sizes on the thermal stability of the adjoining intact base pairs in a duplex DNA chain is studied in a lattice model of Poly(dG) · Poly(dC). We find that for a Y-shaped fork configuration the thermal fluctuation at the fork is so enhanced that the life time of the adjoining base pair is much smaller than the 1 millisecond time scale associated with helicase separation of a base pair in some systems. Our analysis indicates that thermal fluctuational base pair opening may be of importance in facilitating the enzyme unwinding process during chain elongation of a replicating DNA. It is most likely that the thermal fluctuational opening of the base pair at the junction of a replicating fork is fast enough so that a DNA unwinding enzyme can encounter an unstacked base pair with reasonable probability. This conclusion can explain several experimental observations regarding the temporal relationship between ATP hydrolysis by accessory proteins and primer elongation by a holoenzyme complex in ssDNA. We also discuss a mechanism by which the energy associated with ATP hydrolysis may enhance the thermal driven base opening mechanism.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

DNA binding and helicase actions of mouse MCM4/6/7 helicase

Helicases play central roles in initiation and elongation of DNA replication. We previously reported that helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine-rich single-stranded DNA. Here, we examined its substrate preference and helicase actions using various synthetic DNAs. On a bubble substrate, Mcm4/6/7 makes symmetric dual contacts with the 5′-proximal 25 nt single-stranded segments adjacent to the branch points, presumably generating double hexamers. Loss of thymine residues from one single-strand results in significant decrease of unwinding efficacy, suggesting that concurrent bidirectional unwinding by a single double hexameric Mcm4/6/7 may play a role in efficient unwinding of the bubble. Mcm4/6/7 binds and unwinds various fork and extension structures carrying a single-stranded 3′-tail DNA. The extent of helicase activation depends on the sequence context of the 3′-tail, and the maximum level is achieved by DNA with 50% or more thymine content. Strand displacement by Mcm4/6/7 is inhibited, as the GC content of the duplex region increases. Replacement of cytosine–guanine pairs with cytosine–inosine pairs in the duplex restored unwinding, suggesting that mammalian Mcm4/6/7 helicase has difficulties in unwinding stably base-paired duplex. Taken together, these findings reveal important features on activation and substrate preference of the eukaryotic replicative helicase.