BackgroundThe genome of Helicobacter pylori, an oncogenic bacterium in the human stomach, rapidly evolves and shows wide geographical divergence. The high incidence of stomach cancer in East Asia might be related to bacterial genotype. We used newly developed comparative methods to follow the evolution of East Asian H. pylori genomes using 20 complete genome sequences from Japanese, Korean, Amerind, European, and West African strains.ResultsA phylogenetic tree of concatenated well-defined core genes supported divergence of the East Asian lineage (hspEAsia; Japanese and Korean) from the European lineage ancestor, and then from the Amerind lineage ancestor. Phylogenetic profiling revealed a large difference in the repertoire of outer membrane proteins (including oipA, hopMN, babABC, sabAB and vacA-2) through gene loss, gain, and mutation. All known functions associated with molybdenum, a rare element essential to nearly all organisms that catalyzes two-electron-transfer oxidation-reduction reactions, appeared to be inactivated. Two pathways linking acetyl~CoA and acetate appeared intact in some Japanese strains. Phylogenetic analysis revealed greater divergence between the East Asian (hspEAsia) and the European (hpEurope) genomes in proteins in host interaction, specifically virulence factors (tipα), outer membrane proteins, and lipopolysaccharide synthesis (human Lewis antigen mimicry) enzymes. Divergence was also seen in proteins in electron transfer and translation fidelity (miaA, tilS), a DNA recombinase/exonuclease that recognizes genome identity (addA), and DNA/RNA hybrid nucleases (rnhAB). Positively selected amino acid changes between hspEAsia and hpEurope were mapped to products of cagA, vacA, homC (outer membrane protein), sotB (sugar transport), and a translation fidelity factor (miaA). Large divergence was seen in genes related to antibiotics: frxA (metronidazole resistance), def (peptide deformylase, drug target), and ftsA (actin-like, drug target).ConclusionsThese results demonstrate dramatic genome evolution within a species, especially in likely host interaction genes. The East Asian strains appear to differ greatly from the European strains in electron transfer and redox reactions. These findings also suggest a model of adaptive evolution through proteome diversification and selection through modulation of translational fidelity. The results define H. pylori East Asian lineages and provide essential information for understanding their pathogenesis and designing drugs and therapies that target them.

The RecF pathway of Escherichia coli is important for recombinational repair of DNA breaks and gaps. Here we reconstitute in vitro a seven-protein reaction that recapitulates early steps of dsDNA break repair using purified RecA, RecF, RecO, RecR, RecQ, RecJ, and SSB proteins, components of the RecF system. Their combined action results in processing of linear dsDNA and its homologous pairing with supercoiled DNA. RecA, RecO, RecR, and RecJ are essential for joint molecule formation, whereas SSB and RecF are stimulatory. This reconstituted system reveals an unexpected essential function for RecJ exonuclease: the capability to resect duplex DNA. RecQ helicase stimulates this processing, but also disrupts joint molecules. RecO and RecR have two indispensable functions: They mediate exchange of RecA for SSB to form the RecA nucleoprotein filament, and act with RecF to load RecA onto the SSB-ssDNA complex at processed ssDNA-dsDNA junctions. The RecF pathway has many parallels with recombinational repair in eukaryotes.[Keywords: DSB repair; Rad52 group; RecA; RecF; RecQ; recombination] Supplemental material is available at http://www.genesdev.org.

In Escherichia coli, chi (5'-GCTGGTGG-3') is a recombination hotspot recognized by the RecBCD enzyme. Recognition of chi reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by chi interaction has been "ejection" of the RecD motor from the holoenzyme at chi. To test this proposal, we visualized individual RecBCD molecules labeled via RecD with a fluorescent nanoparticle. We could directly see these labeled, single molecules of RecBCD moving at up to 1835 bp/s (approximately 0.6 microm/s). Those enzymes translocated to chi, paused, and continued at reduced velocity, without loss of RecD. We conclude that chi interaction induces a conformational change, resulting from binding of chi to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/ Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.A type II restriction enzyme, such as R.EcoRI, will make a double-stranded break at a specific sequence on DNA (49). A cognate modification enzyme (M.EcoRI) can methylate the same sequence and protect it from restriction cleavage. The genes involved are tightly linked and form a type II restrictionmodification (RM) gene complex. Type II RM gene complexes will attack unmodified foreign DNA such as bacteriophage DNA but not the modified DNA of the cells where they reside. They have been considered to function as bacterial tools against invasion by foreign DNA.We found that elimination of type II RM gene complexes from bacterial cells by a competing genetic element inhibits cell growth (43,44). Our experiments suggested the following course of events after a cell has lost a type II RM gene complex. In the descendants of the cell that have lost the type II RM gene complex, the number of molecules of the modification enzyme will decrease with each cell division. Eventually, the capacity of the enzyme to modify the many sites needed to protect the newly replicated chromosomes from the remaining pool of restriction enzyme will become inadequate. Chromosomal DNA will then be cleaved at the unmodified sites, and the cells will be killed. This is reminiscent of postsegregational cell killing mechanisms, which have been shown to contribute to the stable maintenance of plasmids (11,12,21). Indeed, linkage of several type II RM gene complexes stabilizes plasmids (29,32,43...

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virusinfected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.A type II restriction endonuclease makes a double-strand break within or near a specific recognition sequence in duplex DNA. A cognate modification enzyme methylates the recognition sequence to protect it from the cleavage (1, 2). It is widely accepted that the evolution and maintenance of restriction-modification (RM) systems have been driven by the protection from foreign DNA that they afford to cells. The RM systems do protect cells from infection with some viruses by cleaving their DNA (for example, see ref.3) and are likely to be responsible both for the evolution of antirestriction mechanisms and for the paucity of some restriction sites in certain viruses and plasmids (4).Recent experimental and theoretical analyses (5, 6), however, seem to us to bring into question the efficacy of virusmediated selection for RM systems. Defense by RM systems is short-lived because invading viral DNA will occasionally escape restriction and will become modified, thus affording protection from restriction to itself and its descendants (5, 6). Bacteria will more likely develop other, longer-lasting means of resistance to viruses, such as alterations in the receptor required for infection (5, 6). Although RM systems can provide bacteria with advantage when they are invading new habitats full of phages, it is not clear whether such colonization selection is realistic under natural conditions (5, 6).It is also unclear whether the above "cellular defense" hypothesis can account for the following properties of type II RM systems (1, 2). (i) Their individual high specificity and collective wide diversity in the sequence recognition. (ii) The tight linkage of cognate restriction and modification ...

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Effect of UV-B radiation on the fatty acid composition of the marine phytoplankter Tetraselmis sp.: relationship to cellular pigments Joaquim I. ~o e s l *~, Nobuhiko H a n d a l , Satoru ~a g u c h i~, Takeo ~a m a '

MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.