Homologous Pairing and Dna Strand-Exchange Proteins

Kowalczykowski, Stephen C.; Eggleston, Angela K.

doi:10.1146/annurev.bi.63.070194.005015

Cited by 325 publications

(225 citation statements)

References 89 publications

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“…Inactivation of either the recF or recB gene had no or only a small effect on the deletion frequency, but the combined inactivation of both recF and recB almost completely abolished RecAdependent recombination between diverged DNA repeats (Table 1). Substrates for the RecFOR pathway, single-strand breaks and gaps, can be converted to double-strand breaks that are processed by the RecBCD pathway, whereas the reverse is not possible (22). Therefore, it may be concluded that single-strand breaks and gaps are the dominant substrates for recombination initiation in the wild-type background, whereas a minority of recombination events is initiated by double-strand breaks.…”

Section: Construction and Characterization Of The Recombination Assaymentioning

confidence: 99%

The frequency and structure of recombinant products is determined by the cellular level of MutL

Elez

Radman

Matić

2007

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

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The presence of repeated DNA sequences is a genomic liability, because interrepeat recombination can result in chromosomal rearrangements. The mismatch repair system prevents recombination between nonidentical repeats, but the mechanism of antirecombination has not been established. Although the MutS protein binds to base pair mismatches in heteroduplex DNA, the role of the MutL protein in preventing recombination is unknown. In a screen designed to identify new cellular functions that suppress deletion formation involving nonidentical DNA repeats, we isolated a mutL mutant having a separation-of-function phenotype. The mutant showed an increased frequency of deletions but not of mutations. The split phenotype is due to a decreased MutL level, indicating that recombination, but not replication editing, is highly sensitive to MutL level. By altering the MutL level, we found that the frequency of deletion-generating recombination is inversely related to the amount of cellular MutL. DNA sequence analysis of the recombined repeats shows that the tolerance of base pair mismatches in heteroduplex DNA is also inversely correlated with MutL level. Unlike recombination, correction of misincorporation errors by mismatch repair is insensitive to fluctuations in MutL level. Overproduction of MutS does not affect either of these phenotypes, suggesting that, unlike MutL, MutS is not limiting for mismatch repair activities. These results indicate that MutL (i) determines effective DNA homology in recombination processes and (ii) fine tunes the process of deletion formation involving repeated, diverged DNA sequences.deletions ͉ DNA repeats ͉ mismatch repair ͉ recombination ͉ replication P rokaryotic genomes are riddled with DNA sequence repeats, which are found in intergenic regions, genes, and transposable elements (1). Repeats are even more abundant in eukaryotic genomes (2), and at least 34% of the human genome consists of such sequences (3). The presence of dispersed repeated DNA sequences is a genetic liability because interrepeat recombination can cause deletions, duplications, inversions or translocations, depending on the configuration and orientation of the repeat units. In humans, recombination between repeats is at the origin of disease-causing deletions, such as ␣-thalassemias, Duchenne muscular dystrophy, and familial hypercholesterolemia (4-7).Recombination between repeats is constantly initiated by DNA damage and/or DNA replication blockage that results from exogenous and endogenous genomic insults. Therefore, natural selection for genome stability has resulted in the emergence of mechanisms that prevent interrepeat recombination. Newly arising repeats are identical at the DNA sequence level, but established repeats have usually diverged (8). The degree and distribution of sequence divergence are structural parameters that influence recombination between repeats because they affect the activity of recombination enzymes and determine whether the antirecombinogenic activity of mismatch repair (MMR) proteins is trig...

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Section: Construction and Characterization Of The Recombination Assaymentioning

confidence: 99%

The frequency and structure of recombinant products is determined by the cellular level of MutL

Elez

Radman

Matić

2007

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

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“…At the biochemical level, RecA protein catalyses the invasion of duplex DNA by a homologous single DNA strand. This process leads to the displacement of a single strand in the duplex by the invading strand (Kowalczykowski et al 1994). The genome of the yeast Saccharomyces cerevisiae encodes four proteins that are related to the Escherichia coli recA protein: RAD51, RAD55, RAD57 and DMC1 (Aboussekhra et al 1992;Basile et al 1992;Shinohara et al 1992;Bishop 1994;Lovett 1994).…”

Section: Introductionmentioning

confidence: 99%

Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination

et al. 1997

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“…However, optimal rates and yields in RecA-mediated DNA strand exchange require an additional 6 -8 mM of "free" Mg 2ϩ (4 -6). Some of this magnesium ion is associated with the DNA, but the DNA concentration in most experiments is on the order of a few (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) M in total nucleotides (and backbone phosphate) and thus could not complex more than a small fraction of the available magnesium ion. Lower magnesium concentrations, more stoichiometric with the added ATP, are sufficient for primary DNA binding.…”

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confidence: 99%

“…The RecA protein has a small C-terminal domain extending from residue 270 to the protein terminus at residue 352, the function of which has not been fully explored (1,13). The last 24 amino acid residues of the RecA protein are disordered in the published RecA protein crystal structures (14 -16).…”

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Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Lusetti

Shaw

Cox

2003

Journal of Biological Chemistry

122

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Optimal conditions for RecA protein-mediated DNA strand exchange include 6 -8 mM Mg 2؉ 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 Mg 2؉ ) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although singlestranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg 2؉ 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 the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg 2؉ and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg 2؉ 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.The RecA protein of Escherichia coli plays a central role in the processes of homologous DNA recombination and DNA repair. RecA is a DNA-dependent ATPase that catalyzes an in vitro DNA strand exchange reaction between single-stranded (ssDNA) 1 and homologous double-stranded DNA (dsDNA) molecules. The DNA strand exchange reaction takes place in several stages (Fig. 1). The RecA protein forms a nucleoprotein filament that completely encompasses the circular ssDNA. This filament then aligns the bound single strand with a homologous duplex DNA to form a DNA pairing intermediate often referred to as a joint molecule. 1000 base pairs of DNA can be aligned and exchanged in a joint molecule under the empirically defined optimal reaction conditions, which typically include 1-3 mM ATP and about 10 mM magnesium ion. All steps to this point, including the formation of joint molecules, require ATP but not ATP hydrolysis. ATP hydrolysis is needed only to complete the late stages of strand exchange of long DNA substrates, often derived from bacteriophage DNAs. Whereas DNA pairing, leading to joint molecule formation, can occur at either end of a linear duplex, the subsequent and ATP hydrolysisdependent extension of the nascently paired regions is unidirectional, proceeding 5Ј to 3Ј relative to ssDNA initially bound in the filament. Thus, exchange proceeds in one direction along the linear duplex, and joints formed at the "wrong" end in the pairing phase are eliminated. In a DNA strand exchange involving quite long DNAs that leads to nicked circular product formation, the ends of the duplex where the exchange begins and ends are referred to as proximal and distal, respectively ( Fig. 1) (1, 2).Examination of the conditions for an optimal RecA prot...

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Homologous Pairing and Dna Strand-Exchange Proteins

Cited by 325 publications

References 89 publications

The frequency and structure of recombinant products is determined by the cellular level of MutL

The frequency and structure of recombinant products is determined by the cellular level of MutL

Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

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