Nuclease activity is essential for RecBCD recombination in Escherichia coli

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double-stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of chi recombination hot-spots. These paradoxical roles are accommodated by chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that chi converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5' --> 3' exonuclease activity is essential for recombination by RecBCD at chi recombination hot-spots and at dsDNA ends in recD mutants. In the absence of RecB-dependent nuclease function, recombination becomes entirely dependent on the 5' --> 3' single-stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).     

Genetic Dissection of the Biochemical Activities of Recbcd Enzyme

RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.     

Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase

Helicases unwind dsDNA during replication, repair and recombination in an ATP-dependent reaction. The mechanism for helicase activity can be studied using oligonucleotide substrates to measure formation of single-stranded (ss) DNA from double-stranded (ds) DNA. This assay provides an 'all-or-nothing' readout because partially unwound intermediates are not detected. We have determined conditions under which an intermediate in the reaction cycle of Dda helicase can be detected by trapping a partially unwound substrate. The appearance of this intermediate supports a model in which each ssDNA product interacts with the helicase after unwinding has occurred. Kinetic analysis indicates that the intermediate appears during a slow step in the reaction cycle that is flanked by faster steps for unwinding. These observations demonstrate a complex mechanism containing nonuniform steps for a monomeric helicase. The potential biological significance of such a mechanism is discussed.     

DNA Structures Generated during Recombination Initiated by Mismatch Repair of Uv-Irradiated Nonreplicating Phage DNA in Escherichia Coli: Requirements for Helicase, Exonucleases, and Recf and Recbcd Functions

During infection of homoimmune Escherichia coli lysogens (``repressed infections'), undamaged non-replicating λ phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities--UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease--drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggests that the exonucleases also play other roles in generation and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria in which recombination is low--UvrD(-), ExoI(-), ExoVII(-), or RecJ(-)--displays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut(+) UvrD(+) Exo(+)) generate single-stranded regions in nonreplicating UV-irradiated DNA. Both recF and recB recC mutations strikingly reduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.     

大肠杆菌RecQ解旋酶的表达纯化和生物学活性研究

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达,并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性. 体外诱导表达获得纯度达90% 以上并具有高活性的大肠杆菌重组RecQ解旋酶,其可溶性好;经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性. 较之双链DNA（dsDNA）,大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合( P<0.01 ),但与长度不同的dsDNA的结合特性有差异(P<0.01)而与ssDNA没有差异(P>0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P<0.05);大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P<0.01). 这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制,并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助.     

甲磺酸培氟沙星抑制大肠杆菌RecQ解旋酶的体外DNA结合、解链和ATPase活性

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,参与DNA复制、修复、重组、转录及维持端粒稳定等细胞代谢过程,在维持染色体稳定性与完整性中起着重要作用．甲磺酸培氟沙星(pefloxacin mesylate,PFM)是一种新型氟喹诺酮类抗菌药物,对一些革兰氏阴性菌具有明显的杀菌效果,临床上已广泛使用．本研究利用荧光偏振、自由磷检测技术研究PFM对大肠杆菌RecQ解旋酶的DNA结合活性、解链活性、ATPase活性的影响．结果表明,低浓度PFM可促进大肠杆菌RecQ解旋酶与ssDNA、dsDNA结合,达到一定量后PFM则抑制酶与DNA底物的结合,这种影响与DNA底物有关;PFM对RecQ解旋酶的DNA解链活性和ATP酶活性都具有抑制作用,但其抑制的效果有极显著差异(P<0.01)：比较PFM对两种活性抑制的Ci值(对解链活性抑制的Ci值为(1.5±0.2) μmol/L,对ATP酶活性抑制的Ci值为(0.010±0.005) μmol/L)可知,PFM对大肠杆菌RecQ解旋酶ATPase活性的抑制强于其解链活性. 这些结果可为研究以DNA解旋酶为药物靶标的分子机理奠定相关理论基础．     

The C terminus of the AddA subunit of the Bacillus subtilis ATP-dependent DNase is required for the ATP-dependent exonuclease activity but not for the helicase activity.

Comparison of subunit AddA of the Bacillus subtilis AddAB enzyme, subunit RecB of the Escherichia coli RecBCD enzyme, and subunit RecB of the Haemophilus influenzae RecBCD enzyme revealed several regions of homology. Whereas the first seven regions are common among helicases, the two C-terminally located regions are unique for RecB of E. coli and H. influenzae and AddA. Deletion of the C-terminal region resulted in the production of an enzyme which showed moderately impaired levels of ATP-dependent helicase activity, whereas the ATP-dependent exonuclease activity was completely destroyed. The mutant enzyme was almost completely capable of complementing E. coli recBCD and B. subtilis addAB strains with respect to DNA repair and homologous recombination. These results strongly suggest that at least part of the C-terminal region of the AddA protein is indispensable for exonuclease activity and that, in contrast to the exonuclease activity, the helicase activity of the addAB gene product is important for DNA repair and homologous recombination.     

Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3' to 5' DNA helicase

The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene. Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway. The Mph1 protein contains the seven conserved motifs of the superfamily 2 (SF2) family of nucleic acid unwinding enzymes. Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination. To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity. We show that Mph1 has a robust ATPase function that requires single-stranded DNA for activation. Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1. We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3' to 5' polarity with respect to the DNA strand on which this protein translocates. The DNA helicase activity of Mph1 is enhanced by the heterotrimeric single-stranded DNA binding protein replication protein A. These results, thus, establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance.     

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.     

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.     

Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II–VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I–VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2–7 fold) in all enzyme variants tested.     

Biochemical and kinetic characterization of the DNA helicase and exonuclease activities of werner syndrome protein

The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration (1 mm) of Mg(2+) and ATP with a K(m) of 140 microm for the ATP-Mg(2+) complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg(2+) concentration. Mn(2+) and Ni(2+) substituted for Mg(2+) as a cofactor for WRN helicase, whereas Fe(2+) or Cu(2+) (10 microm) profoundly inhibited WRN unwinding in the presence of Mg(2+).Zn(2+) (100 microm) was preferred over Mg(2+) as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn(2+) strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn(2+) binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1:1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding.     

Adjacent residues in the E1 initiator beta-hairpin define different roles of the beta-hairpin in Ori melting, helicase loading, and helicase activity

We have analyzed two residues in the helicase domain of the E1 initiator protein. These residues are part of a highly conserved structural motif, the beta-hairpin, which is present in the helicase domain of all papovavirus initiator proteins. These proteins are unique in their ability to transition from local template melting activity to unwinding. We demonstrate that the beta-hairpin has two functions. First, it is the tool used by the E1 double trimer (DT) to pry open and melt double-stranded DNA. Second, it is required for the unwinding activity of the hexameric E1 helicase. The fact that the same structural element, but not the same residues, contacts both dsDNA in the DT for melting and ssDNA in the double hexamer (DH) for helicase activity provides a link between local origin melting and DNA helicase activity and suggests how the transition between these two states comes about.     

Residues within the conserved helicase motifs of UL9, the origin-binding protein of herpes simplex virus-1, are essential for helicase activity but not for dimerization or origin binding activity

UL9, an essential gene for herpes simplex virus type 1 (HSV-1) DNA replication, exhibits helicase and origin DNA binding activities. It has been hypothesized that UL9 binds and unwinds the HSV-1 origin of replication, creating a replication bubble and promoting the assembly of the viral replication machinery; however, direct confirmation of this hypothesis has not been possible. Based on the presence of conserved helicase motifs, UL9 has been classified as a superfamily II helicase. Mutations in conserved residues of the helicase motifs I-VI of UL9 have been isolated, and most of them fail to complement a UL9 null virus in vivo (Martinez R., Shao L., and Weller S. (1992) J. Virol. 66, 6735-6746). In addition, mutants in motifs I, II, and VI were found to be transdominant (Malik, A. K., and Weller, S. K. (1996) J. Virol. 70, 7859-7866). Here we present the characterization of the biochemical properties of the UL9 helicase motif mutants. We report that mutations in motifs I-IV and VI affect the ATPase activity, and all but the motif III mutation completely abolish the helicase activity. In addition, mutations in these motifs do not interfere with UL9 dimerization or the ability of UL9 to bind the HSV-1 origin of replication. Based on the similarity of the helicase motif sequences between UL9 and UvrB, another superfamily II member with helicase-like activity, we were able to map the UL9 mutations on the structure of the UvrB protein and provide an explanation for the observed phenotypes. Our results indicate that the helicase function of UL9 is indispensable for viral replication, supporting the hypothesis that UL9 is essential for unwinding the HSV-1 origin of replication in vivo. Furthermore, the data presented provide insights into the mechanism of transdominance of the UL9 helicase motif mutants.     

A single nuclease active site of the Escherichia coli RecBCD enzyme catalyzes single-stranded DNA degradation in both directions

The RecBCD enzyme of Escherichia coli is an ATP-dependent DNA exonuclease and a helicase. Its exonuclease activity is subject to regulation by an octameric nucleotide sequence called chi. In this study, site-directed mutations were made in the carboxyl-terminal nuclease domain of the RecB subunit, and their effects on RecBCD's enzymatic activities were investigated. Mutation of two amino acid residues, Asp(1067) and Lys(1082), abolished nuclease activity on both single- and double-stranded DNA. Together with Asp(1080), these residues compose a motif that is similar to one shown to form the active site of several restriction endonucleases. The nuclease reactions catalyzed by the RecBCD enzyme should therefore follow the same mechanism as these restriction endonucleases. Furthermore, the mutant enzymes were unable to produce chi-specific fragments that are thought to result from the 3'-5' and 5'-3' single-stranded exonuclease activities of the enzyme during its reaction with chi-containing double-stranded DNA. The results show that the nuclease active site in the RecB C-terminal 30-kDa domain is the universal nuclease active site of RecBCD that is responsible for DNA degradation in both directions during the reaction with double-stranded DNA. A novel explanation for the observed nuclease polarity switch and RecBCD-DNA interaction is offered.     

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.