The C Terminus of the Escherichia coli RecA Protein Modulates the DNA Binding Competition with Single-stranded DNA-binding Protein

Eggler, Aimee L.; Lusetti, Shelley L.; Cox, Michael M.

doi:10.1074/jbc.m212920200

Cited by 92 publications

(102 citation statements)

References 61 publications

(46 reference statements)

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“…DNA maintenance and repair is a complex process. The ability to regulate the response has been achieved in many organisms by temporal and specialized association and recruitment of protein factors (8,9,17,21,23,25,26). By removing the carboxyl terminus, an undefined role in recruitment by DdrA protein could have been lost.…”

Section: Discussionmentioning

confidence: 99%

The Stable, Functional Core of DdrA from Deinococcus radiodurans R1 Does Not Restore Radioresistance In Vivo

2008

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DdrA protein binds to and protects 3 DNA ends and is essential for preserving the genome integrity of Deinococcus radiodurans following treatment by gamma radiation in an environment lacking nutrients. Limited proteolysis was used to identify a stable and functional protein core, designated DdrA157, consisting of the first 157 residues of the protein. In vitro, the biochemical differences between wild-type and mutant proteins were modest. DdrA exhibits a strong bias in binding DNA with 3 extensions but not with 5 extensions. The mutant DdrA157 exhibited a greater affinity for 5 DNA ends but still bound to 3 ends more readily. However, when we replaced the wild-type ddrA gene with the mutant gene for ddrA157, the resulting D. radiodurans strain became almost as sensitive to gamma radiation as the ddrA knockout strain. These results suggest that while the stable protein core DdrA157 is functional for DNA binding and protection assays in vitro, the carboxyl terminus is required for important functions in vivo. The C terminus may therefore be required for protein or DNA interactions or possibly as a regulatory region for DNA binding or activities not yet identified.

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Section: Discussionmentioning

confidence: 99%

The Stable, Functional Core of DdrA from Deinococcus radiodurans R1 Does Not Restore Radioresistance In Vivo

2008

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“…Biochemically speaking, binding of RecA protein to ssDNA occurs in two steps (nucleation and filament extension), and the presence of SSB protein on ssDNA inhibits the nucleation step in RecA filament formation (32). The RecA730 mutant protein is able to achieve a nucleation step without the help of the RecA loading mediators RecFOR, since RecA730 competes with SSB protein for ssDNA better than wt RecA protein (7,18,19). This biochemical property of the mutant RecA730 protein is in agreement with the findings of genetic studies, i.e., the suppression of UV sensitivity of recFOR mutants by the recA730 allele (39,40) and the fact that the recA730 mutant exhibits high cSOS expression (8,35,44).…”

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confidence: 99%

“…The mutant RecA730 (E38K) protein can be loaded onto ssDNA without the help of loading mediators: RecBCD or RecFOR proteins (7,18). RecA loading by the RecBCD enzyme is well coordinated with the helicase and nuclease functions of the same enzyme and occurs immediately on nascent ssDNA.…”

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Genetic Requirements for High Constitutive SOS Expression inrecA730Mutants of Escherichia coli

2011

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The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5-3 exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a ␤-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5-3 exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo.The RecA protein is a central component of the recombination machinery which is required for double-strand break (DSB) repair and for producing genetic variation during conjugation in bacteria and meiosis in eukaryotes. An additional role of the RecA protein is in the induction of an SOS response. In order to exhibit its biological functions, the RecA protein must be in the form of a RecA-single-stranded DNA (ssDNA) filament (RecA filament). The RecA filament is formed after processing of DNA damage, i.e., DSBs and single-stranded gaps (SSGs). In wild-type (wt) Escherichia coli strains, DSBs are processed into RecA filaments by the RecBCD pathway of recombination, whereas SSGs utilize the RecF recombination pathway (16,17). DSBs can also be processed into RecA filaments by the RecF recombination pathway when the RecBCD enzyme is missing or is inactive, as is the case in a mutant recBC sbcBC(D) strain with multiple mutations (17). Three essential enzymatic activities are required for the processing of DSBs and subsequent RecA filament formation: helicase, 5Ј-3Ј exonuclease, and RecA loading onto ssDNA. After interaction with a Chi site (GCTGGT GG), the RecBCD enzyme exhibits all of these activities, whereas within the RecF recombination machinery, the pa...

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“…In addition, the initiation proteins (such as RecBCD and RecFOR) might interact directly with the RecA protein during the filament nucleation process, altering RecA conformation so that the C-terminus is no longer inhibitory. These data sets suggest that the DNA binding regulation ability of RecA and/or its ability to compete with SSB would allow RecA to gain access to SSB-coated DNA only at the appropriate time, such as after a replication fork stalls, and that the C-terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament, thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations (Eggler et al, 2003;Lusetti et al, 2003aLusetti et al, , 2003b. In MsRecA, the absence of the last 25 amino acids from the C-terminal domain (a region rich in negatively charged amino acids) could have implications for its functions since regulatory mechanisms, such as those described for EcRecA, can be affected.…”

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confidence: 99%

“…The terminal residues 329-352 are mostly highly negatively charged and form a tail that appears disordered in the crystal structure. The presence of these residues may regulate the direct binding of RecA to DNA through electrostatic repulsion of the DNA phosphate chain (Tateishi et al, 1992;Eggler et al, 2003). Since RecA is highly conserved, the aspects described above for EcRecA could be used for studies comprising other organisms.…”

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A model for the RecA protein of Mycoplasma synoviae

et al. 2007

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In this work, we predict a structural model for the RecA protein from M. synoviae (MsRecA) by theoretical homology modeling and evaluate the occurrence of polymorphisms in this protein within several isolates of this species. The structural model suggested for MsRecA conserves the main domains present in MtRecA and EcRecA. The L1 and L2 regions showed six and three amino acid substitutions, respectively, which apparently do not affect the conformation and function of MsRecA. The C-terminal domain is shorter than that found in EcRecA and MtRecA, which may increase its capacity to bind dsDNA and displace SSB, compensating the absence of recombination initiation enzymes. The MS59 isolate RecA sequence showed one polymorphism which does not affect its functions since these belong to the same physical-chemical group.

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The C Terminus of the Escherichia coli RecA Protein Modulates the DNA Binding Competition with Single-stranded DNA-binding Protein

Cited by 92 publications

References 61 publications

The Stable, Functional Core of DdrA from Deinococcus radiodurans R1 Does Not Restore Radioresistance In Vivo

The Stable, Functional Core of DdrA from Deinococcus radiodurans R1 Does Not Restore Radioresistance In Vivo

Genetic Requirements for High Constitutive SOS Expression inrecA730Mutants of Escherichia coli

A model for the RecA protein of Mycoplasma synoviae

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