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Michael M. Cox - One of the best experts on this subject based on the ideXlab platform.
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regulation of deinococcus radiodurans RecA Protein function via modulation of active and inactive nucleoProtein filament states
2013Co-Authors: Khanh V Ngo, Sindhu Chittenipattu, Eileen T Molzberger, Michael M. CoxAbstract:The RecA Protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA Protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 °C, the optimal growth temperature, and at physiological pH 7.5, DrRecA Protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA-binding Protein. The ATPase function of DrRecA nucleoProtein filaments thus exists in an inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA Proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required) or by addition of volume exclusion agents. Single-stranded DNA-binding Protein plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate Proteins in Escherichia coli. The data suggest a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans. Background: The RecA Protein of Deinococcus radiodurans is essential for extreme radiation resistance. Results: A nucleoProtein state of the DrRecA Protein exists that is inactive with respect to ATP hydrolysis. SSB is essential for DrRecA-mediated DNA strand exchange. Conclusion: There are at least two functional states of DrRecA filaments bound to DNA. Significance: A potential new mode of regulation is revealed for DrRecA.
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investigating deinococcus radiodurans RecA Protein filament formation on double stranded dna by a real time single molecule approach
2011Co-Authors: Hsinfang Hsu, Sindhu Chittenipattu, Khanh V Ngo, Michael M. CoxAbstract:With the aid of an efficient, precise, and almost error-free DNA repair system, Deinococcus radiodurans can survive hundreds of double strand breaks inflicted by high doses of irradiation or desiccation. The RecA of Deinococcus radiodurans (DrRecA) plays a central role both in the early phase of repair by an extended synthesis-dependent strand annealing process and in the later more general homologous recombination phase. Both roles likely require DrRecA filament formation on duplex DNA. We have developed single-molecule tethered particle motion (TPM) experiments to study the assembly dynamics of RecA Proteins on individual duplex DNA molecules by observing changes in DNA tether length resulting from RecA binding. We demonstrate that DrRecA nucleation on dsDNA is much faster than Escherichia coli (Ec) RecA Protein, but the extension is slower. This combination of attributes would tend to increase the number and decrease the length of DrRecA filaments relative to those of EcRecA, a feature that may reflect the requirement to repair hundreds of genomic double strand breaks concurrently in irradiated Deinococcus cells.
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purification and characterization of the RecA Protein from neisseria gonorrhoeae
2011Co-Authors: Elizabeth A Stohl, Michael M. Cox, Marielle C Gruenig, Steven H SeifertAbstract:The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The RecA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecANg), we overexpressed and purified the RecANg and SSBNg Proteins and compared their activities to those of the well-characterized E. coli RecA and SSB Proteins in vitro. We observed that RecANg promoted more strand exchange at early time points than RecAEc through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA Protein characterized to date. Further analysis of this robust ATPase activity revealed that RecANg is more efficient at displacing SSB from ssDNA and that RecANg shows higher ATPase activity during strand exchange than RecAEc. Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecAEc catalyzed more strand exchange through a 100 bp heterologous insert, but that RecANg catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecANg than in RecAEc. This difference may explain the unusually high ATPase activity observed for RecANg with the strand exchange activity between RecANg and RecAEc being more similar.
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motoring along with the bacterial RecA Protein
2007Co-Authors: Michael M. CoxAbstract:The recombinases of the RecA family are often viewed only as DNA-pairing Proteins - they bind to one DNA segment, align it with homologous sequences in another DNA segment, promote an exchange of DNA strands and then dissociate. To a first approximation, this description seems to fit the eukaryotic (Rad51 and Dmc1) and archaeal (RadA) RecA homologues. However, the bacterial RecA Protein does much more, coupling ATP hydrolysis with DNA-strand exchange in a manner that greatly expands its repertoire of activities. This article explores the Protein activities and experimental results that have identified RecA as a motor Protein.
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regulation of bacterial RecA Protein function
2007Co-Authors: Michael M. CoxAbstract:The RecA Protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the RecA gene is regulated within the SOS response. The activity of the RecA Protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other Proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD Proteins. The SSB Protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF Proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX Protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI Protein stabilizes RecA filaments. The RdgC Protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB Protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these Proteins function in a network that determines where and how RecA functions. Additional regulatory Proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.
Stephen C Kowalczykowski - One of the best experts on this subject based on the ideXlab platform.
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recfor Proteins target RecA Protein to a dna gap with either dna or rna at the 5 terminus implication for repair of stalled replication forks
2012Co-Authors: Katsumi Morimatsu, Yun Wu, Stephen C KowalczykowskiAbstract:Abstract The repair of single-stranded gaps in duplex DNA by homologous recombination requires the Proteins of the RecF pathway. The assembly of RecA Protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding Protein is accelerated by the RecF, RecO, and RecR (RecFOR) Proteins. Here, we show the RecFOR Proteins specifically target RecA Protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF Protein, in the presence of the RecOR Proteins, to the junction of ssDNA and dsDNA within a gap is 1–2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR Proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoProtein filament on ssDNA for 1000–2000 nucleotides downstream (5′ → 3′) of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR Proteins also recognize RNA at the 5′-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks.
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single molecule analysis of a red fluorescent RecA Protein reveals a defect in nucleoProtein filament nucleation that relates to its reduced biological functions
2009Co-Authors: Naofumi Handa, Steven J. Sandler, Ichiro Amitani, Nathan Gumlaw, Stephen C KowalczykowskiAbstract:Abstract Fluorescent fusion Proteins are exceedingly useful for monitoring Protein localization in situ or visualizing Protein behavior at the single molecule level. Unfortunately, some Proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA Protein (RecA803 Protein) to monomeric red fluorescent Protein (mRFP1) to produce a functional Protein (RecA-RFP) that is suitable for in vivo and in vitro analysis. In vivo, the RecA-RFP partially restores UV resistance, conjugational recombination, and SOS induction to RecA− cells. In vitro, the purified RecA-RFP Protein forms a nucleoProtein filament whose kcat for single-stranded DNA-dependent ATPase activity is reduced ∼3-fold relative to wild-type Protein, and which is largely inhibited by single-stranded DNA-binding Protein. However, RecA Protein is also a dATPase; dATP supports RecA-RFP nucleoProtein filament formation in the presence of single-stranded DNA-binding Protein. Furthermore, as for the wild-type Protein, the activities of RecA-RFP are further enhanced by shifting the pH to 6.2. As a consequence, RecA-RFP is proficient for DNA strand exchange with dATP or at lower pH. Finally, using single molecule visualization, RecA-RFP was seen to assemble into a continuous filament on duplex DNA, and to extend the DNA ∼1.7-fold. Consistent with its attenuated activities, RecA-RFP nucleates onto double-stranded DNA ∼3-fold more slowly than the wild-type Protein, but still requires ∼3 monomers to form the rate-limited nucleus needed for filament assembly. Thus, RecA-RFP reveals that its attenuated biological functions correlate with a reduced frequency of nucleoProtein filament nucleation at the single molecule level.
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translocation by the recb motor is an absolute requirement for χ recognition and RecA Protein loading by recbcd enzyme
2005Co-Authors: Maria Spies, Mark S Dillingham, Stephen C KowalczykowskiAbstract:Abstract RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. The enzyme is driven by two motor subunits, RecB and RecD, translocating on opposite single-strands of the DNA duplex. Here we provide evidence that, although both motor subunits can support the translocation activity for the enzyme, the activity of the RecB subunit is necessary for proper function of the enzyme both in vivo and in vitro. We demonstrate that the RecBCDK177Q enzyme, in which RecD helicase is disabled by mutation of the ATPase active site, complements recBCD deletion in vivo and displays all of the enzymatic activities that are characteristic of the wild-type enzyme in vitro. These include helicase and nuclease activities and the abilities to recognize the recombination hotspot χ and to coordinate the loading of RecA Protein onto the ssDNA it produces. In contrast, the RecBK29QCD enzyme, carrying a mutation in the ATPase site of RecB helicase, fails to complement recBCD deletion in vivo. We further show that even though RecBK29QCD enzyme displays helicase and nuclease activities, its inability to translocate along the 3′-terminated strand results in the failure to recognize χ and to load RecA Protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to χ, whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.
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recfor Proteins load RecA Protein onto gapped dna to accelerate dna strand exchange a universal step of recombinational repair
2003Co-Authors: Katsumi Morimatsu, Stephen C KowalczykowskiAbstract:Abstract Genetic evidence suggests that the RecF, RecO, and RecR (RecFOR) Proteins participate in a common step of DNA recombination and repair, yet the biochemical event requiring collaboration of all three Proteins is unknown. Here, we show that the concerted action of the RecFOR complex directs the loading of RecA Protein specifically onto gapped DNA that is coated with single-stranded DNA binding (SSB) Protein, thereby accelerating DNA strand exchange. The RecFOR complex recognizes the junction between the ssDNA and dsDNA regions and requires a base-paired 5′ terminus at the junction. Thus, the RecFOR complex is a structure-specific mediator that targets recombinational repair to ssDNA-dsDNA junctions. This reaction reconstitutes the initial steps of recombinational gapped DNA repair and uncovers an event also common to the repair of ssDNA-tailed intermediates of dsDNA-break repair. We propose that the behavior of the RecFOR Proteins is mimicked by functional counterparts that exist in all organisms.
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facilitated loading of RecA Protein is essential to recombination by recbcd enzyme
2000Co-Authors: Deana A Arnold, Stephen C KowalczykowskiAbstract:Abstract Although the RecB2109CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3′-terminal single-stranded DNA overhang that is an ideal substrate for RecA Protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the χ-modified wild-type RecBCD enzyme. However, we further show that the RecB2109CD enzyme is unable to coordinate the loading of RecA Protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA Protein by the χ-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.
Ross B. Inman - One of the best experts on this subject based on the ideXlab platform.
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An SOS inhibitor that binds to free RecA Protein: the PsiB Protein.
2009Co-Authors: Vessela Petrova, Julia C. Drees, Sindhu Chitteni-pattu, Ross B. Inman, Michael CoxAbstract:The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB Protein inhibits all activities of the RecA Protein, including DNA binding, DNA strand exchange, and LexA Protein cleavage. The Proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoProtein filament. However, PsiB binds to RecA Protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoProtein filaments on DNA.
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complementation of one RecA Protein point mutation by another evidence for trans catalysis of atp hydrolysis
2006Co-Authors: Julia M Cox, Sindhu Chittenipattu, Ross B. Inman, Stephen N Abbott, Michael M. CoxAbstract:The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoProtein filament. The K248R and E96D single mutant Proteins of the Escherichia coli RecA Protein each bind to DNA and form nucleoProtein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant Proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the Proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA Protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type Protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoProtein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.
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inhibition of RecA Protein function by the rdgc Protein from escherichia coli
2006Co-Authors: Julia C. Drees, Sindhu Chittenipattu, Ross B. Inman, Darrell R Mccaslin, Michael M. CoxAbstract:The Escherichia coli RdgC Protein is a potential negative regulator of RecA function. RdgC inhibits RecA Protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI Protein. RdgC Protein can inhibit DNA strand exchange catalyzed by RecA nucleoProtein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoProtein filaments. RdgC Protein binds to single-stranded and double-stranded DNA, and the Protein can be visualized on DNA using electron microscopy. RdgC Protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.
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The DinI Protein Stabilizes RecA Protein Filaments
2004Co-Authors: Shelley L. Lusetti, Ross B. Inman, Oleg N. Voloshin, R. Daniel Camerini-otero, Michael CoxAbstract:When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI Protein was present at very high concentrations, generally superstoichiometric, relative to the RecA Protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA Protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.
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c terminal deletions of the escherichia coli RecA Protein characterization of in vivo and in vitro effects
2003Co-Authors: Shelley L. Lusetti, Ross B. Inman, Alberto I Roca, Elizabeth A Wood, Christopher D Fleming, Michael J Modica, Joshua Korth, Lily Abbott, David W Dwyer, Michael M. CoxAbstract:A set of C-terminal deletion mutants of the RecA Protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C.In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type Protein promotes a proficient reaction. However, at the same time, the deletion of 13–17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type Protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the Protein. These interactions affect the pK a of key groups involved in DNA strand exchange as well as the direct binding of RecA Protein to duplex DNA.
Shelley L. Lusetti - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of RecA Protein by the Escherichia coli RecX Protein: modulation by the RecA C terminus and filament functional state.
2004Co-Authors: Julia C. Drees, Shelley L. LusettiAbstract:Abstract The RecX Protein is a potent inhibitor of RecA activities. We identified several factors that affect RecX-RecA interaction. The interaction is enhanced by the RecA C terminus and by significant concentrations of free Mg2+ ion. The interaction is also enhanced by an N-terminal His6 tag on the RecX Protein. We conclude that RecX Protein interacts most effectively with a RecA functional state designated Ao and that the RecA C terminus has a role in modulating the interaction. We further identified a C-terminal point mutation in RecA Protein (E343K) that significantly alters the interaction between RecA and RecX Proteins.
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The DinI Protein Stabilizes RecA Protein Filaments
2004Co-Authors: Shelley L. Lusetti, Ross B. Inman, Oleg N. Voloshin, R. Daniel Camerini-otero, Michael CoxAbstract:When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI Protein was present at very high concentrations, generally superstoichiometric, relative to the RecA Protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA Protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.
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the c terminus of the escherichia coli RecA Protein modulates the dna binding competition with single stranded dna binding Protein
2003Co-Authors: Aimee L Eggler, Shelley L. Lusetti, Michael M. CoxAbstract:The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding Protein (SSB). The capacity of RecA Protein to displace SSB is dramatically enhanced in RecA Proteins with C-terminal deletions. The displacement of SSB by RecA Protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA Protein relative to the full-length Protein. The C-terminal deletion mutants also more readily displace yeast replication Protein A than does the full-length Protein. Thus, the RecA Protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecAΔC17 also has an enhanced capacity relative to wild-type RecA Protein to bind ssDNA containing secondary structure. Added Mg2+ enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication Protein A. The overall binding of RecAΔC17 mutant Protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecAΔC17/E38K displaces SSB somewhat better than either individual mutant Protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.
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c terminal deletions of the escherichia coli RecA Protein characterization of in vivo and in vitro effects
2003Co-Authors: Shelley L. Lusetti, Ross B. Inman, Alberto I Roca, Elizabeth A Wood, Christopher D Fleming, Michael J Modica, Joshua Korth, Lily Abbott, David W Dwyer, Michael M. CoxAbstract:A set of C-terminal deletion mutants of the RecA Protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C.In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type Protein promotes a proficient reaction. However, at the same time, the deletion of 13–17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type Protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the Protein. These interactions affect the pK a of key groups involved in DNA strand exchange as well as the direct binding of RecA Protein to duplex DNA.
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Magnesium ion-dependent activation of the RecA Protein involves the C terminus.
2003Co-Authors: Shelley L. Lusetti, Jeffrey J. Shaw, Michael M. CoxAbstract:Abstract Optimal conditions for RecA Protein-mediated DNA strand exchange include 6–8 mm Mg2+ in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA Protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a “closed” (low Mg2+) conformation of a RecA nucleoProtein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg2+ results in an “open” conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at theEscherichia coli RecA C terminus eliminates a measurable requirement for excess Mg2+ and permits efficient DNA pairing and exchange similar to that seen with the wild-type Protein at high Mg2+ levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.
Michael Cox - One of the best experts on this subject based on the ideXlab platform.
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regulation of deinococcus radiodurans RecA Protein function via modulation of active and inactive nucleoProtein filament states
2013Co-Authors: Khanh V Ngo, Sindhu Chittenipattu, Eileen T Molzberger, Michael CoxAbstract:Abstract The RecA Protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA Protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 °C, the optimal growth temperature, and at physiological pH 7.5, DrRecA Protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA-binding Protein. The ATPase function of DrRecA nucleoProtein filaments thus exists in an inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA Proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required) or by addition of volume exclusion agents. Single-stranded DNA-binding Protein plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate Proteins in Escherichia coli. The data suggest a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans.
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An SOS inhibitor that binds to free RecA Protein: the PsiB Protein.
2009Co-Authors: Vessela Petrova, Julia C. Drees, Sindhu Chitteni-pattu, Ross B. Inman, Michael CoxAbstract:The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB Protein inhibits all activities of the RecA Protein, including DNA binding, DNA strand exchange, and LexA Protein cleavage. The Proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoProtein filament. However, PsiB binds to RecA Protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoProtein filaments on DNA.
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The DinI Protein Stabilizes RecA Protein Filaments
2004Co-Authors: Shelley L. Lusetti, Ross B. Inman, Oleg N. Voloshin, R. Daniel Camerini-otero, Michael CoxAbstract:When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI Protein was present at very high concentrations, generally superstoichiometric, relative to the RecA Protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA Protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.
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RecA Protein filaments disassemble in the 5' to 3' direction on single-stranded DNA.
2001Co-Authors: Julie M. Bork, Michael Cox, Ross B. InmanAbstract:Abstract RecA Protein forms filaments on both single- and double-stranded DNA. Several studies confirm that filament extension occurs in the 5′ to 3′ direction on single-stranded DNA. These filaments also disassemble in an end-dependent fashion, and several indirect observations suggest that the disassembly occurs on the end opposite to that at which assembly occurs. By labeling the 5′ end of single-stranded DNA with a segment of duplex DNA, we demonstrate unambiguously that RecA filaments disassemble uniquely in the 5′ to 3′ direction.
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RecA Protein FILAMENTS : END-DEPENDENT DISSOCIATION FROM SSDNA AND STABILIZATION BY RECO AND RECR ProteinS
1997Co-Authors: Qun Shan, Ross B. Inman, Julie M. Bork, Brian L. Webb, Michael CoxAbstract:RecA Protein filaments formed on circular (ssDNA) in the presence of ssDNA binding Protein (SSB) are generally stable as long as ATP is regenerated. On linear ssDNA, stable RecA filaments are believed to be formed by nucleation at random sites on the DNA followed by filament extension in the 5' to 3' direction. This view must now be enlarged as we demonstrate that RecA filaments formed on linear ssDNA are subject to a previously undetected end-dependent disassembly process. RecA Protein slowly dissociates from one filament end and is replaced by SSB. The results are most consistent with disassembly from the filament end nearest the 5' end of the DNA. The bound SSB prevents re-formation of the RecA filaments, rendering the dissociation largely irreversible. The dissociation requires ATP hydrolysis. Disassembly is not observed when the pH is lowered to 6.3 or when dATP replaces ATP. Disassembly is not observed even with ATP when both the RecO and RecR Proteins are present in the initial reaction mixture. When the RecO and RecR Proteins are added after most of the RecA Protein has already dissociated, RecA Protein filaments re-form after a short lag. The newly formed filaments contain an amount of RecA Protein and exhibit an ATP hydrolysis rate comparable to that observed when the RecO and RecR Proteins are included in the initial reaction mixture. The RecO and RecR Proteins thereby stabilize RecA filaments even at the 5' ends of ssDNA, a fact which should affect the recombination potential of 5' ends relative to 3' ends. The location and length of RecA filaments involved in recombinational DNA repair is dictated by both the assembly and disassembly processes, as well as by the presence or absence of a variety of other Proteins that can modulate either process.
