Effect of the Streptococcus pneumoniae MmsA protein on the RecA protein-promoted three-strand exchange reaction. Implications for the mechanism of transformational recombination

Streptococcus pneumoniae is a naturally transformable bacterium that is able to incorporate DNA from its environment into its own chromosome. This process, known as transformational recombination, is dependent in part on the mmsA gene, which encodes a protein having a sequence that is 40% identical to that of the Escherichia coli RecG protein, a junction-specific DNA helicase believed to be involved in the branch migration of recombinational intermediates. We have developed an expression system for the MmsA protein and have purified the MmsA protein to more than 99% homogeneity. The MmsA protein has DNA-dependent ATP hydrolysis and DNA junction-helicase activities that are similar to those of the E. coli RecG protein. The effect of the MmsA protein on the S. pneumoniae RecA protein-promoted three-strand exchange reaction was also investigated. In the standard direction (circular single-stranded (ss) DNA + linear double-stranded (ds) DNA --> linear ssDNA + nicked circular dsDNA), the MmsA protein appears to promote the branch migration of partially exchanged intermediates in a direction opposite of the RecA protein, resulting in a nearly complete inhibition of the overall strand exchange reaction. In the reverse direction (linear ssDNA + nicked circular dsDNA --> circular ssDNA + linear dsDNA), however, the MmsA protein appears to facilitate the conversion of partially exchanged intermediates into fully exchanged products, leading to a pronounced stimulation of the overall reaction. These results are discussed in terms of the molecular mechanism of transformational recombination.     

Stimulation of the Streptococcus pneumoniae RecA protein-promoted three-strand exchange reaction by the competence-specific SsbB protein

The effect of the transformational competence-specific Streptococcus pneumoniae single-stranded DNA binding protein, SpSsbB, on the ATP-dependent three-strand exchange activity of the SpRecA protein was investigated. Although SpRecA exhibited only a trace level of strand exchange activity in the absence of SpSsbB, an extensive strand exchange reaction was observed when SpSsbB was added to the reaction solution after SpRecA. A more limited strand exchange reaction was observed, however, when SpSsbB was added to the reaction solution before SpRecA. This dependence on the order of addition, together with additional DNA-dependent ATP hydrolysis experiments, indicated that the mechanism of stimulation may involve the postsynaptic binding of SpSsbB to the displaced linear single-stranded DNA reaction product. When dATP was provided in place of ATP as the nucleotide cofactor (to suppress a potentially inhibitory effect of SpSsbB on the interaction of SpRecA with the circular ssDNA reaction substrate), the stimulatory effect of SpSsbB on the strand exchange reaction was apparent regardless of the order in which it was added to the reaction solution. These findings suggest that SpSsbB may be able to facilitate SpRecA-promoted DNA recombination reactions during natural transformation in S. pneumoniae.     

The pgdA gene encodes for a peptidoglycan N-acetylglucosamine deacetylase in Streptococcus pneumoniae

Analytical work on the fractionation of the glycan strands of Streptococcus pneumoniae cell wall has led to the observation that an unusually high proportion of hexosamine units (over 80% of the glucosamine and 10% of the muramic acid residues) was not N-acetylated, explaining the resistance of the peptidoglycan to the hydrolytic action of lysozyme, a muramidase that cleaves in the glycan backbone. A gene, pgdA, was identified as encoding for the peptidoglycan N-acetylglucosamine deacetylase A with amino acid sequence similarity to fungal chitin deacetylases and rhizobial NodB chitooligosaccharide deacetylases. Pneumococci in which pgdA was inactivated by insertion duplication mutagenesis produced fully N-acetylated glycan and became hypersensitive to exogenous lysozyme in the stationary phase of growth. The pgdA gene may contribute to pneumococcal virulence by providing protection against host lysozyme, which is known to accumulate in high concentrations at infection sites.     

The licC gene of Streptococcus pneumoniae encodes a CTP:phosphocholine cytidylyltransferase

The licC gene product of Streptococcus pneumoniae was expressed and characterized. LicC is a nucleoside triphosphate transferase family member and possesses CTP:phosphocholine cytidylyltransferase activity. Phosphoethanolamine is a poor substrate. The LicC protein plays a role in the biosynthesis of the phosphocholine-derivatized cell wall constituents that are critical for cell separation and pathogenesis.     

Inhibition of DNA repair by neighbouring mismatched bases in Streptococcus pneumoniae

A set of pneumococcal strains containing immediately adjacent or nearby double mutations at the amiA locus, conferring resistance to amethopterin, has been isolated by oligonucleotide site-specific mutagenesis. Repair of these double mutations has been measured by transformation of wild-type strains with DNA extracted from these strains. In several transformations we have observed an inhibition of repair by neighbouring mismatches. This inhibition ranges from mild to severe depending upon the interfering mismatch. Unrepaired mismatches can strongly inhibit repair of an adjacent repairable mutation. This suggests that the repair-complex proteins attach not only to repairable mismatches but also to some mismatches known to escape the repair system.     

A locus of group A Streptococcus involved in invasive disease and DNA transfer

Group A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic-tag-lengths-transposon-mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft-tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil, streptococcal invasion locus. sil contains at least five genes (silA-E) and is highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two-component system whereas silD and silE encode two putative ABC transporters. silC is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of silC was restored when injected together with the avirulent emm14-deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm14-deletion mutant and mutants of sil occurred also in vitro, at a frequency of approximately 10-8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.     

The Streptococcus pneumoniae beta-galactosidase is a surface protein

The beta-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous beta-galactosidase activity. The results are consistent with beta-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.     

DNA repair protein involved in heart and blood development

Apurinic/apyrimidinic endonuclease 1, a key enzyme in repairing abasic sites in DNA, is an embryonic lethal in mice. We are examining its role in embryogenesis in zebra fish. Zebra fish contain two genomic copies (zfAPEX1a and zfAPEX1b) with identical coding sequences. zfAPEX1b lacks introns. Recombinant protein (ZAP1) is highly homologous with and has the same enzymatic properties as its human orthologue. ZAP1 is highly expressed throughout development. Embryos microinjected with morpholino oligonucleotide (MO) targeting the translation start site die at approximately the midblastula transition (MBT) without apoptosis. They are rescued with mRNA for human wild-type APEX1 but not for APEX1 encoding endonuclease-defective protein. Rescued embryos develop dysmorphic hearts, pericardial edema, few erythrocytes, small eyes, and abnormal notochords. Although the hearts in rescued embryos form defective loops ranging from no loop to one that is abnormally shaped, cardiac myosin (cmlc2) is present and contraction occurs. Embryos microinjected with MO targeting zfAPEX1a intron-exon junctions also pass the MBT with similar abnormalities. We conclude that AP endonuclease 1 is involved in both repairing DNA and regulating specific early stages of embryonic development.     

Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins.

Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker 'A' motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW-K141R, inhibits replication from a T4 origin at which persistent RNA-DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double-strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA-DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW-K141R fusion, displays ssDNA-dependent ATPase activity and is able to unwind a branched DNA substrate.     

The histone-like protein HU binds specifically to DNA recombination and repair intermediates

The heterodimeric HU protein associated with the Escherichia coli nucleoid shares some properties with histones and HMG proteins. HU binds DNA junctions and DNA containing a nick much more avidly than double-stranded (ds-) DNA. Cells lacking HU are extremely sensitive to gamma irradiation and we wondered how HU could play a role in maintaining the integrity of the bacterial chromosome. We show that HU binds with high affinity to DNA repair and recombination intermediates, including DNA invasions, DNA overhangs and DNA forks. The DNA structural motif that HU specifically recognizes in all these structures consists of a ds-DNA module joined to a second module containing either ds- or single-stranded (ss-) DNA. The two modules rotate freely relative to one another. Binding specificity results from the simultaneous interaction of HU with these two modules: HU arms bind the ds-DNA module whereas the HU body contacts the 'variable' module containing either ds- or ss-DNA. Both structural motifs are recognized by HU at least 1000-fold more avidly than duplex DNA.     

Homologous recombination is involved in repair of chromium-induced DNA damage in mammalian cells

Chromium is a potent human carcinogen, probably because of its well-documented genotoxic effects. Chromate (Cr[VI]) causes a wide range of DNA lesions, including DNA crosslinks and strand breaks, presumably due to the direct and indirect effects of DNA oxidation. Homologous recombination repair (HRR) is important for error-free repair of lesions occurring at replication forks. Here, we show that HR deficient cell lines irs1SF and V-C8, deficient in XRCC3 and BRCA2, respectively, are hypersensitive to Cr[VI], implicating this repair pathway in repair of Cr[VI] damage. Furthermore, we find that Cr[VI] causes DNA double-strand breaks and triggers both Rad51 foci formation and induction of HRR. Collectively, these data suggest that HRR is important in repair of Cr[VI]-induced DNA damage. In addition, we find that ERCC1, XRCC1 and DNA-PKcs defective cells are hypersensitive to Cr[VI], indicating that several repair pathways cooperate in repairing Cr[VI]-induced DNA damage.     

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.     

The sir locus of Escherichia coli: a gene involved in SOS-independent repair of mitomycin C-induced DNA damage

A Mud1 (lac Apr) insertion has been isolated in a delta (lac)recA+ lexA3(Ind-)rpoB87 gyrA87 mutant of Escherichia coli resulting in a decrease in mitomycin C tolerance and an increase in post-mitomycin C DNA degradation. The mitomycin C sensitivity of the insertion mutant is not further increased by substituting either the rpoB87 or the gyrA mutation by the respective wild-type alleles. However, when both rpoB87 and gyrA87 mutations are replaced by rpoB+ and gyrA+ the strain becomes hypersensitive to mitomycin C. Inactivation of recA in the insertion mutant has no effect on its mitomycin C sensitivity provided both rpoB87 and gyrA87 are present. When either or both of the mutations is/are replaced by the wild-type allele inactivation of recA renders the strain hypersensitive to mitomycin C. The locus of Mud1 (lac Apr) insertion, designated sir (SOS-independent repair), has been mapped between 57 and 61 min on the E. coli linkage map. Expression of the sir gene seems to be constitutive and not enhanced by mitomycin C. These results are discussed in relation to the SOS-independent repair of mitomycin C-induced DNA damage reported earlier.     

The recombination, repair and modification of DNA

This article is an overview of the author's involvement in theoretical and experimental research on genetic recombination and DNA repair, and also on the enzymic modification of cytosine in DNA to 5-methyl cytosine. It includes the history of the discovery of the central intermediate in genetic recombination at the DNA level, and the repair of mismatched bases. These explain the major features of genetic fine structure. The first repair and recombination defective mutants in any eukaryote were isolated in the smut fungus Ustilago maydis. The hypothesis that DNA methylation has a role in gene expression in higher organism is now supported by abundant evidence. Direct evidence that gene silencing in mammalian cells is causally related to DNA methylation has been obtained.     

A homolog of ScRAD5 is involved in DNA repair and homologous recombination in Arabidopsis

Rad5 is the key component in the Rad5-dependent error-free branch of postreplication repair in yeast (Saccharomyces cerevisiae). Rad5 is a member of the Snf2 ATPase/helicase family, possessing as a characteristic feature, a RING-finger domain embedded in the Snf2-helicase domain and a HIRAN domain. Yeast mutants are sensitive to DNA-damaging agents and reveal differences in homologous recombination. By sequence comparisons we were able to identify two homologs (AtRAD5a and AtRAD5b) in the Arabidopsis thaliana genome, sharing about 30% identity and 45% similarity to yeast Rad5. AtRad5a and AtRad5b have the same kind of domain organization with a higher degree of similarity to each other than to ScRad5. Surprisingly, both genes differ in function: whereas two independent mutants of Atrad5a are hypersensitive to the cross-linking agents mitomycin C and cis-platin and to a lesser extent to the methylating agent, methyl methane sulfonate, the Atrad5b mutants did not exhibit any sensitivity to all DNA-damaging agents tested. An Atrad5a/Atrad5b double mutant resembles the sensitivity phenotype of the Atrad5a single mutants. Moreover, in contrast to Atrad5b, the two Atrad5a mutants are deficient in homologous recombination after treatment with the double-strand break-inducing agent bleomycin. Our results suggest that the RAD5-dependent error-free branch of postreplication repair is conserved between yeast and plants, and that AtRad5a might be functionally homologous to ScRad5.