In many eubacteria, coexpression of recX with recA is essential for attenuation of the deleterious effects of recA overexpression; however, the molecular mechanism has remained enigmatic. Here T he past several years have seen a considerable progress in our understanding of the central role of Escherichia coli RecA protein in homologous recombination, DNA repair, restoration of stalled replication forks, induction of SOS response, and mutagenesis (1, 2). The much-studied homologous recombination process in vitro is the three-strand exchange reaction between circular single-and linear double-stranded DNA (1-3). In this model, the reaction proceeds in three sequential phases: (i) The presynaptic polymerization of RecA protein on singlestranded DNA; (ii) synapsis, the homologous alignment of nucleoprotein filament with linear double-stranded DNA; and (iii) unidirectional strand exchange (1-3). The mechanistic aspects of homologous recombination promoted by the prototype E. coli RecA protein may arguably be the best understood, but understanding its counterparts from other organisms will be essential to establish the generality of the phenomenon. To this end, we have described the biochemical characterization and x-ray structure of M. tuberculosis RecA (4-6).In some eubacteria, recX is located on the same coding strand downstream of recA (7). In Streptomyces lividans, Mycobacterium smegmatis, Mycobacterium tuberculosis, Pseudomonas aeruginosa, or Thiobacillus ferrooxidans, the ORFs of recA and recX overlap and the two genes are cotranscribed (7-12). It is known that overexpression of recA in recX mutants of S. lividans, M. smegmatis, or P. aeruginosa, but not mutant RecA, lead to induction of deleterious effects (8, 10, 13). However, the molecular mechanisms by which recX attenuates the deleterious effects induced by recA overexpression has remained unknown. Using M. tuberculosis as a model, we explored the mechanism by which RecA is regulated by RecX. Here, we show that RecX interacts directly with RecA in vitro and in vivo resulting in suppression of ATPase and strand exchange, processes that are central to homologous recombination. The negative regulation of RecA by RecX implies that RecX might act as an antirecombinase to quell inappropriate recombinational repair during normal DNA metabolism. (14) and their concentrations determined as described (15). Negatively supercoiled (form I) and circular single-stranded M13 DNA (ssDNA) was prepared as described (16). The concentrations are expressed in moles of nucleotide residues. Materials and Methods Purification of RecX. E. coli BL21(DE3)[pLysS] strain harboringM. tuberculosis recX gene on plasmid pET15b was cultured in 1 liter of LB medium containing 50 g͞ml ampicillin and 34 g͞ml chloramphenicol at 37°C. At mid-exponential phase (A 600 ϭ 0.4), recX expression was induced by adding isopropyl ␤-Dthiogalactoside (IPTG) to a final concentration of 0.5 mM and incubated for 4 h. All subsequent steps were performed at 4°C unless indicated otherwise. Cell paste (8 g) was...

Mycobacterium tuberculosis recA harbors an intervening sequence in its open reading frame, presumed to encode an endonuclease (PI-MtuI) required for intein homing in inteinless recA allele. Although the proteinsplicing ability of PI-MtuI has been characterized, the identification of its putative endonuclease activity has remained elusive. To investigate whether PI-MtuI possesses endonuclease activity, recA intervening sequence was cloned, overexpressed, and purified to homogeneity. Here we show that PI-MtuI bound both single-and double-stranded DNA with similar affinity but failed to cleave DNA in the absence of cofactors. Significantly, PI-MtuI nicked supercoiled DNA in the presence of alternative cofactors but required both Mn 2؉ and ATP to generate linear double-stranded DNA. We observed that PI-MtuI was able to inflict a staggered double-strand break 24 bp upstream of the insertion site in the inteinless recA allele. Similar to a few homing endonucleases, DNA cleavage by PI-MtuI was specific with an exceptionally long cleavage site spanning 22 bp. The kinetic mechanism of PI-MtuI promoted cleavage supports a sequential rather than concerted pathway of strand cleavage with the formation of nicked double-stranded DNA as an intermediate. Together, these results reveal that RecA intein is a novel Mn 2؉ -ATP-dependent doublestrand specific endonuclease, which is likely to be important for homing process in vivo.The prototype Escherichia coli RecA protein plays a central role in homologous recombination, DNA repair, restoration of stalled replication forks and SOS response (reviewed in Refs.

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the "homing" process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

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