An Interaction between the Escherichia coli RecF and RecR Proteins Dependent on ATP and Double-stranded DNA

Webb, Brian; Cox, Michael M.; Inman, Ross B.

doi:10.1074/jbc.270.52.31397

Cited by 77 publications

(147 citation statements)

References 37 publications

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“…An 8-l sample of KaiC protein was diluted 100-fold with 200 mM ammonium acetate͞Hepes (10 mM, pH 7.0)͞10% glycerol, and adsorbed to the Alcianactivated carbon film for 3 min. After washing with the same buffer for 1 min, the sample was stained with 5% uranyl acetate followed by a very brief circular shadowing with platinum (19).…”

Section: Methodsmentioning

confidence: 99%

“…Reaction mixtures were prepared for EM by one of two procedures: (i) Alcian method (positive staining): Samples were prepared by adsorption to Alcian-activated carbon-coated grids as previously described (19). An 8-l sample of KaiC protein was diluted 100-fold with 200 mM ammonium acetate͞Hepes (10 mM, pH 7.0)͞10% glycerol, and adsorbed to the Alcianactivated carbon film for 3 min.…”

Section: Methodsmentioning

confidence: 99%

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Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

Mori

Saveliev

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA͞DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA͞DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.DnaB ͉ RecA ͉ cyanobacteria ͉ Synechococcus

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

Mori

Saveliev

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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139

155

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“…However, the RecR binding sites for the RecO and RecF have not been identified. In addition, E. coli RecR has been reported to form a dimer in solution (15,20), and its structural conformation has not been determined. Previously, we reported the backbone NMR assignments of Thermus thermophilus RecR (ttRecR) and suggested that it forms a symmetric homodimer in solution (23).…”

mentioning

confidence: 99%

“…recombinational DNA repair at replication forks (17,18). The RecF protein itself binds DNA and has a weak ATPase activity (19), both of which are enhanced by RecR binding (20,21). Furthermore, specific loading of the RecA protein onto the dsDNA-ssDNA junction is mediated by the RecF, RecO, and RecR proteins acting in concert, with no interaction between RecF and RecO detected (4).…”

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confidence: 99%

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

Honda

Inoue

Yoshimasu

et al. 2006

Journal of Biological Chemistry

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The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.Maintaining genomic integrity by repairing damaged DNA is crucial for all organisms. DNA damage can arise during normal DNA metabolism such as the introduction of mismatches during replication and the deamination of bases, or it can be caused by exposure to exogenous factors such as ultraviolet radiation, ␥-radiation, and chemical mutagens. Such DNA damage is mostly repaired by base excision repair, nucleotide excision repair, and mismatch repair pathways, based on information provided by the complementary strand in a damaged duplex. However, double-stranded DNA (dsDNA) 2 break and base lesions in single-stranded DNA (ssDNA) gap regions having no complementary strands can also arise, mainly during replication. To cope with this category of DNA damage, organisms have developed recombinational repair pathways that minimize the loss of genetic information by using homologous DNA as a template for repair.RecFOR and/or RecBCD pathways are required to initiate homologous DNA recombination in bacteria (1). The Escherichia coli RecFOR pathway is mainly used for ssDNA gap repair, whereas the RecBCD pathway is responsible for dsDNA break repair. However, the RecFOR pathway can also repair dsDNA breaks, as has been demonstrated in recBC sbcB mutants, which are deficient in the RecBCD pathway (2, 3). In the RecFOR pathway, dsDNA breaks are unwound by the RecQ helicase and processed by the RecJ 5Ј-to 3Ј-exonuclease, and the resulting 3Ј-tailed ssDNA is coated with the ssDNA-binding protein (SSB). The RecF, RecO, and RecR proteins then mediate the loading of RecA protein onto the SSB-coated ssDNA, specifically at junctio...

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“…Reactions were stopped by adding ATP␥S to 1 mM and incubating at 37°C for 5 min. Reactions were spread by a modified adsorption procedure onto carbon films that were activated by either Alcian blue (33) or denatured BSA (as described above), with the following changes. The samples were diluted into 200 mM ammonium acetate, 10 mM HEPES, pH 8.0, and 10% (w/v) glycerol and were adsorbed to carbon films on an EM grid for 3 min.…”

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confidence: 99%

RecA Protein Filaments Disassemble in the 5′ to 3′ Direction on Single-stranded DNA

Bork¹,

Cox

Inman

2001

Journal of Biological Chemistry

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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 singlestranded 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.The active form of the bacterial RecA protein is a filament formed on DNA. RecA has no known activities when it is not part of such a filamentous complex. Further, the action of several other bacterial proteins appears to be directed, at least in part, to the modulation of RecA filament assembly and disassembly on DNA (1-5). An understanding of the formation and disassembly of these filaments is thus critical to a broader understanding of RecA function in recombination processes and to an understanding of the activities of many other recombination proteins.Filament formation generally includes a distinct and ratelimiting nucleation step followed by rapid filament extension. RecA filaments are formed most readily on ssDNA, 1 where nucleation is followed by extension uniquely in the 5Ј to 3Ј direction (1, 6). Filament formation on dsDNA is very slow above pH 7 due to slow nucleation (7,8). On a gapped DNA substrate, RecA will nucleate within the single-stranded gapped region, and the subsequent extension will encompass any adjacent duplex DNA on the 3Ј-proximal end of the gap (1, 6, 9 -11). The RecA protein forms a right-handed helical filament on DNA with visible striations seen using EM (12, 13). Within the filament, bound DNA is highly extended and underwound (12, 14 -18).The bacterial SSB protein modulates many aspects of RecA filament dynamics. Prebound SSB inhibits the nucleation step in RecA filament assembly on ssDNA (19). However, once RecA filament formation is initiated, SSB facilitates the subsequent filament extension by removing secondary structure in the DNA (20). Even though filament nucleation is inhibited by SSB, the same SSB is readily displaced during the extension phase of filament formation. When SSB is overproduced within the Escherichia coli cell, recombination of UV-irradiated bacteriophage DNA is specifically inhibited (21).A net end-dependent disassembly of RecA protein filaments has been observed on dsDNA (11, 22) and ssDNA (1). Net filament disassembly from dsDNA and ssDNA is observed at neutral and higher pH values. On ssDNA, observation of a net disassembly is dependent upon the presence of SSB and ATP (1) and proceeds at a rate of about 60 -70 monomers min Ϫ1 at 37°C. In the EM, filament disassembly on linear singlestranded DNA appears to occur only on one end (1). Because filament extension occurs unambiguously in the 5Ј to 3Ј direction, and because extension followed by recession at the same end seemed unlikely, we previously argued that filament disassembly must b...

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An Interaction between the Escherichia coli RecF and RecR Proteins Dependent on ATP and Double-stranded DNA

Cited by 77 publications

References 37 publications

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

RecA Protein Filaments Disassemble in the 5′ to 3′ Direction on Single-stranded DNA

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