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1.
Appropriate initiation of the strand exchange reaction promoted by RecA protein requires ATP hydrolysis 总被引:2,自引:0,他引:2
The DNA-dependent ATPase activity of the Escherichia coli RecA protein has been recognized for more than two decades. Yet, the role of ATP hydrolysis in the RecA-promoted strand exchange reaction remains unclear. Here, we demonstrate that ATP hydrolysis is required as part of a proofreading process during homology recognition. It enables the RecA-ssDNA complex, after determining that the strand-exchanged duplex is mismatched, to dissociate from the synaptic complex, which allows it to re-initiate the search for a "true" homologous region. Furthermore, the results suggest that when non-homologous sequences are present at the proximal end, ATP hydrolysis is required to allow ssDNA-RecA to reinitiate the strand exchange from an internal homologous region. 相似文献
2.
Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda.
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The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts. 相似文献
3.
Energetics of RecA-mediated recombination reactions. Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle 总被引:11,自引:0,他引:11
We demonstrate that the step of DNA strand exchange during RecA-mediated recombination reaction can occur equally efficiently in the presence or absence of ATP hydrolysis. The polarity of strand exchange is the same when instead of ATP its non-hydrolyzable analog adenosine-5'-O-(3-thiotriphosphate) is used. We show that the ATP dependence of recombination reaction is limited to the post-exchange stages of the reactions. The low DNA affinity state of RecA protomers, induced after ATP hydrolysis, is necessary for the dissociation of RecA-DNA complexes at the end of the reaction. This dissociation of RecA from DNA is necessary for the release of recombinant DNA molecules from the complexes formed with RecA and for the recycling of RecA protomers for another round of the recombination reaction. 相似文献
4.
The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them. Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51. However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different. When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51. For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis. Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, I.e. the reaction can get stuck. Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate. 相似文献
5.
Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides 总被引:8,自引:0,他引:8
K M Woo W J Chung D B Ha A L Goldberg C H Chung 《The Journal of biological chemistry》1989,264(4):2088-2091
Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it. 相似文献
6.
7.
Summary The cloned recA
+ gene of Proteus mirabilis substitutes for a defective RecA protein in Escherichia coli recA
– mutants, and restores recombination, repair and phage induction functions to near normal levels. In a previous report, we described the purification and charactrisation of the recombination activities of the P. mirabilis RecA protein (West et al. 1983b). In this paper, we show that the purified protein catalyses the cleavage of both the Escherichia coli LexA protein and the bacteriophage lambda repressor in vitro. These results provide a direct biochemical basis for the interspecies complementation observed in vivo and suggest that P. mirabilis has an SOS regulatory network similar to that of E. coli. 相似文献
8.
RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli . ATP hydrolysis is known to be important for RecA's roles in recombination and DNA repair. In vitro reactions modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo . The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA–GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA+ , depending on the ratio of the two proteins. RecA4159 was unable to enter RecA+ filaments on DNA or storage structures and was recessive to RecA+ . It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament. 相似文献
9.
The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene. 相似文献
10.
RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski et al., 1990), calling into question the role of ATP hydrolysis in this reaction. We demonstrate here that the ATP gamma S-mediated process is restricted to homologous strand exchange reactions involving three strands. In four-strand exchanges between a gapped duplex circle and a second linear duplex, joint molecules are formed in the gap but are not extended into the four-strand region when ATP gamma S is present. This result provides evidence that one function of ATP hydrolysis in the recA system is to facilitate reciprocal DNA strand exchange involving four strands. Implications with respect to the role of four-stranded pairing intermediates and the mechanistic relationship between three- and four-strand exchange reactions are discussed. 相似文献
11.
DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange 总被引:1,自引:0,他引:1
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PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction. 相似文献
12.
Chanchal DasGupta Anna M. Wu Roger Kahn Richard P. Cunningham Charles M. Radding 《Cell》1981,25(2):507-516
RecA protein makes stable joint molecules from fully duplex DNA and molecules that are partially single-stranded; the latter may be either duplex molecules with an internal gap in one strand or molecules with single-stranded ends. Stable joint molecules form only when the end of at least one strand is in a homologous region. When RecA protein pairs linear duplex molecules and tailed molecules that share the same sequence end to end, the joints, which are located away from the single-stranded tails in most instances, have the electron microscopic appearance associated with the Holliday structure resulting from the reciprocal exchange of strands. The reaction leading to reciprocal strand exchange involves the concerted displacement of a strand from the end of the duplex molecule. These observations support the view that RecA protein makes stable joint molecules only by transferring strands and not by the side-by-side pairing of duplex regions. 相似文献
13.
14.
Remote but significant similarities between the primary and predicted secondary structures of the chloramphenicol acetyltransferases (CAT) and lipoate acyltransferase subunits (LAT, E2) of the 2-oxo acid dehydrogenase complexes, have suggested that both types of enzyme may use similar catalytic mechanisms. Multiple sequence alignments for CAT and LAT have highlighted two conserved motifs that contain the active-site histidine and serine residues of CAT. Site-directed replacement of Ser550 in the E2p subunit (LAT) of the pyruvate dehydrogenase complex of Escherichia coli, deemed to be equivalent to the active-site Ser148 of CAT, supported the CAT-based model of LAT catalysis. The effects of other substitutions were also consistent with the predicted similarity in catalytic mechanism although specific details of active-site geometry may not be conserved. 相似文献
15.
Transcription but not translation is required for EDTA-induced autolysis in Escherichia coli 总被引:1,自引:0,他引:1
Rifampicin, but not chloramphenicol or other inhibitors of translation, inhibited EDTA-induced autolysis in Escherichia coli. Inhibition of EDTA-induced autolysis in E. coli was also observed with nalidixic acid and novobiocin, inhibitors of topoisomerase II. Rifampicin or nalidixic acid-resistant mutants of E. coli were resistant to the inhibitory effect of the respective antibiotic on EDTA-induced autolysis. The implications of these studies in regard to our understanding of the regulation of autolysis in E. coli are discussed. 相似文献
16.
17.
Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation. 总被引:8,自引:4,他引:4
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Motif III is one of the seven protein motifs that are characteristic of superfamily I helicases. To investigate its role in the helicase mechanism we have introduced a variety of mutations at three of the most conserved amino acid residues (Q254, W259 and R260). Biochemical characterisation of the resulting proteins shows that mutation of motif III affects both ATP hydrolysis and single-stranded DNA binding. We propose that amino acid residue Q254 acts as a gamma-phosphate sensor at the nucleotide binding pocket transmitting conformational changes to the DNA binding site, since the nature of the charge on this residue appears to control the degree of coupling between ATPase and helicase activities. Residues W259 and R260 both participate in direct DNA binding interactions that are critical for helicase activity. 相似文献
18.
T Horii 《Biochimie》1991,73(2-3):177-185
The RecA protein of E coli promotes a strand exchange reaction in vitro which appears to be similar to homologous genetic recombination in vivo. A model for the mechanism of strand transfer reaction by RecA protein has been proposed by Howard-Flanders et al based on the assumption that the RecA monomer has two distinctive DNA binding sites both of which can bind to ssDNA as well as dsDNA. Here, I propose an alternative model based on the assumption that RecA monomer has a single domain for binding to a polynucleotide chain with a unique polarity. In addition, the model is based on a few mechanical assumptions that, in the presence of ATP, two RecA molecules form a head to head dimer as the basic binding unit to DNA, and that the binding of RecA protein to a polynucleotide chain induces a structural change of RecA protein that causes a higher state of affinity for another RecA molecule that is expressed as cooperativy. The model explains many of the biochemical capabilities of RecA protein including the polar polymerization of RecA protein on single stranded DNA and polar strand transfer of DNA by the protein as well as the formation of a joint DNA molecule in a paranemic configuration. The model also presents the energetics in the strand transfer reaction. 相似文献
19.
The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed. 相似文献
20.
SecA is an essential ATP-driven motor protein that binds to preproteins and the translocon to promote protein translocation across the eubacterial plasma membrane. Escherichia coli SecA contains seven conserved motifs characteristic of superfamily II of DNA and RNA helicases, and it has been shown previously to possess RNA helicase activity. SecA has also been shown to be an autogenous repressor that binds to its translation initiation region on secM-secA mRNA, thereby blocking and dissociating 30 S ribosomal subunits. Here we show that SecA is an ATP-dependent helicase that unwinds a mimic of the repressor helix of secM-secA mRNA. Mutational analysis of the seven conserved helicase motifs in SecA allowed us to identify mutants that uncouple SecA-dependent protein translocation activity from its helicase activity. Helicase-defective secA mutants displayed normal protein translocation activity and autogenous repression of secA in vivo. Our studies indicate that SecA helicase activity is nonessential and does not appear to be necessary for efficient protein secretion and secA autoregulation. 相似文献