Functional similarities between phage λ Orf and
            <i>Escherichia coli</i>
            RecFOR in initiation of genetic exchange

Maxwell, Karen L.; Reed, Patricia; Zhang, Rongguang; Beasley, Steven; Walmsley, Adrian R.; Curtis, Fiona A.; Joachimiak, Andrej; Edwards, A.M.; Sharples, Gary J.

doi:10.1073/pnas.0503399102

Cited by 21 publications

(44 citation statements)

References 47 publications

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“…An Orfmodified SSB may be a specialized modification that allows RecA to easily displace Beta without interference from SSB. In partial support of this proposal, Maxwell et al (142) found that Orf binds to the E. coli SSB protein.…”

Section: Other λ Functions That Influence the Mechanism Of Red Recombmentioning

confidence: 68%

“…In addition, the study demonstrated that Orf binds to ssDNA. Using dsDNA substrates containing ssDNA gaps, the authors determined that ssDNA binds to a U-shaped cleft on the surface of the protein rather than going through the central channel (142). They suggested that a fluorescence quench observed when Orf binds ssDNA could indicate a conformational change that allows preferential binding of RecA or Beta.…”

Section: Other λ Functions That Influence the Mechanism Of Red Recombmentioning

confidence: 99%

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λ Recombination and Recombineering

Murphy

2016

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The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

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Section: Other λ Functions That Influence the Mechanism Of Red Recombmentioning

confidence: 68%

Section: Other λ Functions That Influence the Mechanism Of Red Recombmentioning

confidence: 99%

λ Recombination and Recombineering

Murphy

2016

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“…Precisely how these three proteins (wileyonlinelibrary.com) DOI:10.1002/jmr.1079 assemble at dsDNA-ssDNA junctions and promote RecA nucleation is unclear. Some of the in vitro properties of Orf resemble those reported for RecFOR, including binding to DNA and a specific interaction with E. coli SSB, although, Orf does not stimulate annealing of complementary ssDNA molecules (Maxwell et al, 2005). The Orf crystal structure reveals an asymmetric homodimer arranged as a toroid (Maxwell et al, 2005).…”

Section: Introductionmentioning

confidence: 78%

“…Some of the in vitro properties of Orf resemble those reported for RecFOR, including binding to DNA and a specific interaction with E. coli SSB, although, Orf does not stimulate annealing of complementary ssDNA molecules (Maxwell et al, 2005). The Orf crystal structure reveals an asymmetric homodimer arranged as a toroid (Maxwell et al, 2005). Bound ssDNA is predicted to occupy a shallow groove across the protein surface rather than threading through the central channel.…”

Section: Introductionmentioning

confidence: 79%

The C‐terminus of the phage λ Orf recombinase is involved in DNA binding

Curtis

Reed

Wilson

et al. 2010

J of Molecular Recognition

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Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.

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“…Proteins that facilitate the formation of the filaments of RecA-class recombinases are called recombination mediator proteins (21). Recombination mediator proteins are as ubiquitous as are the recombinases themselves (21,(23)(24)(25)(26)(27)(28). The E. coli RecF, RecO, and RecR proteins are perhaps the prototypical recombination mediator proteins (12, 21, 29 -32), part of a larger network of bacterial proteins that regulate almost every aspect of RecA function (22,30,33).…”

mentioning

confidence: 99%

RecFOR and RecOR as Distinct RecA Loading Pathways

Sakai

Cox

2009

Journal of Biological Chemistry

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The molecular role of the RecF protein in loading RecA protein onto single-stranded DNA (ssDNA)-binding proteincoated ssDNA has been obscured by the facility with which the RecO and RecR proteins alone perform this function. We now show that RecFOR and RecOR define distinct RecA loading functions that operate optimally in different contexts. RecFOR, but not RecOR, is most effective when RecF(R) is bound near an ssDNA/double-stranded (dsDNA) junction. However, RecF(R) has no enhanced binding affinity for such a junction. RecO and RecR proteins are both required under all conditions in which the RecFOR pathway operates. The RecOR pathway is uniquely distinguished by a required interaction between RecO protein and the ssDNA binding protein C terminus. The RecOR pathway is more efficient for RecA loading onto ssDNA when no proximal dsDNA is available. A merger of new and published results leads to a new model for RecFOR function.Recombination reactions catalyzed by the RecA protein form an integral part of DNA metabolism in Escherichia coli. The major role for recombination in bacteria is the repair of stalled replication forks (1-4). E. coli strains lacking intact recombination systems exhibit sensitivity to DNA damaging agents, hypermutability, and impaired growth rates (5).RecA is found in all eubacteria with the exception of a few endosymbionts (Buchnera sp.) (6 -8). It functions as a helical nucleoprotein filament, formation of which requires ATP and Mg 2ϩ ion. When bound to single-stranded DNA (ssDNA), 2RecA hydrolyzes ATP. The active filament can promote the exchange of DNA strands between homologous DNA molecules in vitro. RecA filament formation occurs in at least two distinct phases (9). Filament nucleation occurs first and involves binding of a few (probably 4 -5 (10)) RecA protomers to DNA. Nucleation is rate-limiting and is followed by rapid filament extension in the 5Ј to 3Ј direction along the DNA (11-14). RecA filament disassembly requires ATP hydrolysis and also occurs in the 5Ј to 3Ј direction along ssDNA (14). Recombinase function is highly regulated in all cells, and the regulation occurs at many levels. The LexA repressor protein controls expression of the recA gene (15, 16). The 17 C-terminal amino acids of RecA function in autoregulation of most protein activities (17-19). Finally, E. coli possesses many additional proteins that function as recombination regulators, with RecA as their target (20 -22). Most of these proteins affect the kinetics of filament formation and disassembly. Proteins that facilitate the formation of the filaments of RecA-class recombinases are called recombination mediator proteins (21). Recombination mediator proteins are as ubiquitous as are the recombinases themselves (21,(23)(24)(25)(26)(27)(28). The E. coli RecF, RecO, and RecR proteins are perhaps the prototypical recombination mediator proteins (12, 21, 29 -32), part of a larger network of bacterial proteins that regulate almost every aspect of RecA function (22,30,33).The ssDNA-binding protein (SSB; 18.2 kDa) ex...

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Functional similarities between phage λ Orf and Escherichia coli RecFOR in initiation of genetic exchange

Cited by 21 publications

References 47 publications

λ Recombination and Recombineering

λ Recombination and Recombineering

The C‐terminus of the phage λ Orf recombinase is involved in DNA binding

RecFOR and RecOR as Distinct RecA Loading Pathways

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