Orientation specificity of the <i>Lactococcus lactis</i> Chi site

Karoui, Meriem El; Schaeffer, M.I.; Biaudet, Véronique; Bolotin, Alexandre; Sorokin, Alexeï; Gruss, Alexandra

doi:10.1046/j.1365-2443.2000.00342.x

Cited by 10 publications

(9 citation statements)

References 48 publications

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“…As expected, very few recombinants were obtained in the ⌬recBCD host, regardless of whether Chi Ll were present on the linear dsDNA ends. Recombination via wild-type RexAB was stimulated 30-fold by the presence of Chi on incoming linear fragments, in keeping with previous results (22,23). In the presence of pRexA ⌬DYK B, recombination was at background levels, further confirming that a change in the RexA nuclease motif results in total inactivation of RexAB biological activities in vivo.…”

Section: Rexbsupporting

confidence: 89%

“…In addition, the Chi Lldependent increase in HMW accumulation shows that the remaining nuclease activity is still attenuated at Chi Ll . Possibly, the RexB subunit degrades just one of the two dsDNA strands from the 5Ј end (i.e., the strand containing the Chi complement) (23). These results confirm the nuclease-defective phenotype of RexB Nuc as seen in the T4g2 test.…”

Section: (See Materials and Methods)supporting

confidence: 67%

“…Gene replacement with linear DNA fragments is stimulated if correctly oriented Chi sites are present in the linear DNA flanking regions of homologies (15,22,23). Using this criterion, it was previously demonstrated that the presence of Chi Ll stimulates homologous recombination (22,23). We examined the ability of mutant RexAB exo/ hel to mediate Chi Ll -stimulated homologous recombination by using linear fragments with or without double Chi Ll sites at the ends ( Fig.…”

Section: Rexbmentioning

confidence: 99%

“…The diversity in DSB repair enzymes may be related to genome plasticity: genome rearrangements are common events that may occur via intrachromosomal transposition, gene duplications and inversions, or entry of exogenous DNA. In L. lactis, there is a 4:1 orientation bias of Chi distribution with respect to the direction of DNA replication (exo/hel recognizes Chi in one orientation) (14,21,23,29,47). Inversion of a large DNA segment could considerably reduce the number of Chi sites available to stimulate repair if a replication fork break occurs in that region (36).…”

Section: Rexbmentioning

confidence: 99%

“…RexAB bears homologues in at least six other gram-positive low-guanine-plus-cytosine-content bacteria as well as in the gram-negative bacterium Porphyromonas gingivalis (determined by BLAST comparison; http://www.ncbi.nlm.nih.gov/BLAST/). As studied in L. lactis or in Bacillus subtilis (AddAB), the two-subunit exo/hel enzymes display biological and/or biochemical activities equivalent to those of RecBCD (i.e., ATPdependent exonuclease, helicase, exonuclease blocking at Chi, and Chi-stimulated recombination; 8,9,10,22,23,28,30,31). RexA and its analogues in other gram-positive bacteria have homology with PcrA helicase (determined by BLAST comparison; http://www.ncbi.nlm.nih.gov/BLAST/), whose mechanism has been deduced from structural determinations (49).…”

mentioning

confidence: 99%

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In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis

et al. 2001

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In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.In bacteria, double-stranded DNA breaks (DSB) are frequent events that may be provoked, for example, by pauses in the replication fork (36, 43). Such genomic disruptions are lethal in the absence of DNA repair. In Escherichia coli DSB repair requires the activity of a large enzyme complex, known as RecBCD, that has ATP-dependent helicase and exonuclease activities (see reference 32 for a review). The enzyme degrades both strands, starting from the DNA break until it reaches an octanucleotide sequence, known as Chi, that attenuates degradation and stimulates recombination (44,46). The enzyme exhibits helicase activity and residual exonuclease activity with an altered polarity after Chi (4, 16); the remaining activity provides a single-stranded DNA substrate for recombination enzymes to mediate repair.Organization of the three-subunit exonuclease/helicase (exo/hel) RecBCD. Structure-functional studies of RecBCD have revealed some of the roles of each subunit. RecB seems to possess two key activities of the enzyme. The N-terminal 929 amino acids (out of 1,180 total) have confirmed ATPase and helicase activities (13,54

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Section: Rexbsupporting

confidence: 89%

Section: (See Materials and Methods)supporting

confidence: 67%

Section: Rexbmentioning

confidence: 99%

Section: Rexbmentioning

confidence: 99%

mentioning

confidence: 99%

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In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis

et al. 2001

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The FtsK Family of DNA Pumps

Demarre

Galli

Barre

2012

Advances in Experimental Medicine and Biology

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Interest for proteins of the FtsK family initially arose from their implication in many primordial processes in which DNA needs to be transported from one cell compartment to another in eubacteria. In the first section of this chapter, we address a list of the cellular functions of the different members of the FtsK family that have been so far studied. Soon after their discovery, interest for the FstK proteins spread because of their unique biochemical properties: most DNA transport systems rely on the assembly of complex multicomponent machines. In contrast, six FtsK proteins are sufficient to assemble into a fast and powerful DNA pump; the pump transports closed circular double stranded DNA molecules without any covalent-bond breakage nor topological alteration; transport is oriented despite the intrinsic symmetrical nature of the double stranded DNA helix and can occur across cell membranes. The different activities required for the oriented transport of DNA across cell compartments are achieved by three separate modules within the FtsK proteins: a DNA translocation module, an orientation module and an anchoring module. In the second part of this chapter, we review the structural and biochemical properties of these different modules.

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Orientation specificity of the Lactococcus lactis Chi site

Cited by 10 publications

References 48 publications

In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis

In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis

The FtsK Family of DNA Pumps

Séquences exceptionnelles dans l'ADN

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