The beta protein of phage λ binds preferentially to an intermediate in DNA renaturation

Karakousis, Giorgos C.; Ye, N; Li, Z; Chiu, Sung-Kay; Reddy, Gurucharan; Radding, Charles M.

doi:10.1006/jmbi.1997.1573

Cited by 77 publications

(107 citation statements)

References 37 publications

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“…Recombineering utilizes the bacteriophage λ generalized recombination Red functions, Exo and Beta, along with the Gam protein to catalyze recombination with linear DNA substrates. Red Exo and Beta are, respectively, a 5'->3' double strand (ds)-exonuclease (Little, 1967) and a single strand (ss)-annealing protein (Karakousis et al, 1998) that together catalyze recombination between short homologies of around 50 bases (Yu et al, 2000). Gam inhibits the E. coli exonuclease RecBCD (Unger et al, 1972) allowing intracellular preservation of linear ds DNA, such as PCR products.…”

Section: Introductionmentioning

confidence: 99%

Multicopy plasmid modification with phage λ Red recombineering

Thomason¹,

Costantino²,

Shaw³

et al. 2007

Plasmid

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Recombineering, in vivo genetic engineering using the bacteriophage λ Red generalized recombination system, was used to create various modifications of a multicopy plasmid derived from pBR322. All genetic modifications possible on the E. coli chromosome and on bacterial artificial chromosomes (BACs) are also possible on multicopy plasmids and are obtained with similar frequencies to their chromosomal counterparts, including creation of point mutations (5-10% unselected frequency), deletions and substitutions. Parental and recombinant plasmids are nearly always present as a mixture following recombination, and circular multimeric plasmid molecules are often generated during the recombineering. Keywordsphage λ Red homologous recombination; multicopy plasmid; plasmid multimers; in vivo genetic engineering

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Section: Introductionmentioning

confidence: 99%

Multicopy plasmid modification with phage λ Red recombineering

Thomason¹,

Costantino²,

Shaw³

et al. 2007

Plasmid

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“…Exo and RecE are 5Ј-3Ј exonucleases that degrade the 5Ј ends of linear duplex DNA, creating 3Ј single-strand (ss) DNA overhangs (8,9). Beta and RecT are single-strand annealing proteins that bind to these ssDNA overhangs and pair them with complementary ssDNA targets (10)(11)(12). Gam inhibits two potent host nucleases, RecBCD and SbcCD (13,14), thereby preventing the degradation of linear double-strand (ds) DNA introduced into the cell.…”

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confidence: 99%

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages

Datta

Costantino

Zhang

et al. 2008

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

176

233

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We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda () bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear doublestrand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria.bacteriophage lambda ͉ Beta ͉ oligonucleotide recombination ͉ RecT ͉ ssDNA annealing proteins R ecombineering is a simple and efficient way to engineer DNA molecules in vivo without the in vitro use of restriction enzymes and DNA ligase (1, 2). In Escherichia coli, it allows genetic modifications, from point mutations to substitutions and deletions. Recombineering is mediated by phage-derived proteins, either the Red proteins of phage (1-3) or RecET from the Rac prophage of E. coli (4), which are particularly efficient in catalyzing homologous recombination between short (Ϸ50 bp) homology segments. The Red recombination functions are encoded by three adjacent genes, gam, bet and exo, in the pL operon (1). RecET functions are encoded by two adjacent genes, recE and recT, present on the cryptic lambdoid Rac prophage found in the genome of many E. coli K-12 strains (5). A variety of studies have concluded that the Exo/Beta and RecE/RecT protein pairs are functionally equivalent although not related at the sequence level (6, 7). Exo and RecE are 5Ј-3Ј exonucleases that degrade the 5Ј ends of linear duplex DNA, creating 3Ј single-strand (ss) DNA overhangs (8, 9). Beta and RecT are single-strand annealing proteins that bind to these ssDNA overhangs and pair them with complementary ssDNA targets (10-12). Gam inhibits two potent host nucleases, RecBCD and SbcCD (13,14), thereby preventing the degradation o...

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“…␤ protein promotes annealing of complementary single strands (7,8) and migration of a single-stranded branch (9). This protein, which does not hydrolyze ATP, binds more strongly to the product of its annealing activity, which may help to explain how it drives branch migration (10), but there are no correlations of structure with function that help us to understand the role of either the rings or the left-handed filaments. RecT protein, the product of a cryptic prophage in E. coli and a functional homolog of ␤ protein, also forms rings and filaments.…”

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confidence: 99%

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

Gupta

Golub

et al. 2001

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

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Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A⅐T base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a ''synaptic pathway'' involving a three-stranded nucleoprotein intermediate, rather than by a ''helicase pathway'' involving the separation and reannealing of DNA strands.T wo kinds of macromolecular protein structures play prominent roles in homologous genetic recombination: toroids and filaments. Among the toroidal structures, the best understanding of the relation of function to structure exists for Escherichia coli RuvB protein, which forms a hexameric ATPase͞helicase. Two such hexameric rings, interacting with RuvA protein, drive the migration of Holliday structures, the 4-fold branched DNA intermediate formed after initial synapsis and processing have occurred. The consequence of this driven migration is the reciprocal exchange of a pair of like strands between two duplex molecules (1). Rings are also formed by the exonuclease of phage (2), the Erf protein of phage P22 (3), and human Rad52 protein (4, 5). On the basis of the crystal structure of toroidal exonuclease, Kovall and Matthews suggested a mechanistic explanation of the high processivity of this enzyme (2).Some recombination proteins form both rings and filaments: the ␤ protein of phage forms large rings and left-handed helical filaments (6). ␤ protein promotes annealing of complementary single strands (7,8) and migration of a single-stranded branch (9). This protein, which does not hydrolyze ATP, binds more strongly to the product of its annealing activity, which may help to explain how it drives branch migration (10), but there are no correlations of structure with function that help us to understand the role of either the rings or the left-handed filaments. RecT protein, the product of a cryptic prophage in E. coli and a functional ...

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The beta protein of phage λ binds preferentially to an intermediate in DNA renaturation

Cited by 77 publications

References 37 publications

Multicopy plasmid modification with phage λ Red recombineering

Multicopy plasmid modification with phage λ Red recombineering

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

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