1 A Recombination Puzzle Solved : Role for New DNA Repair Systems in Helicobacter pylori Diversity / Persistence

1.1 Helicobacter pylori pathogenesis Helicobacter pylori is a gram-negative, slow-growing, microaerophilic, spiral bacterium. It is one of the most common human gastrointestinal pathogens, infecting almost 50% of the world’s population [1]. Peptic ulcer disease is now approached as an infectious disease, and H. pylori is responsible for the majority of duodenal and gastric ulcers [2]. There is strong evidence that H. pylori infection increases the risk of gastric cancer [3], the second most frequent cause of cancer-related death. H. pylori infections are acquired by oral ingestion and is mainly transmitted within families in early childhood [2]. Once colonized, the host can be chronically infected for life, unless H. pylori is eradicated by treatment with antibiotics. H. pylori is highly adapted to its ecologic niche, the human gastric mucosa. The pathogenesis of H. pylori relies on its persistence in surviving a harsh environment, including acidity, peristalsis, and attack by phagocyte cells and their released reactive oxygen species [4]. H. pylori has a unique array of features that permit entry into the mucus, attachment to epithelial cells, evasion of the immune response, and as a result, persistent colonization and transmission. Numerous virulence factors in H. pylori have been extensively studied, including urease, flagella, BabA adhesin, the vacuolating cytotoxin (VacA), and the cag pathogenicity island (cag-PAI) [5]. In addition to its clinical importance, H. pylori has become a model system for persistent host-associated microorganisms [6]. How H. pylori can adapt to, and persist in, the human stomach has become a problem of general interest in both microbial physiology and in pathogenesis areas.

A Recombination Puzzle Solved: Role for New DNA Repair Systems in Helicobacter pylori Diversity/Persistence 5 only portions of them.H. pylori encodes the homologues of all four members of the nucleotide excision repair (NER) pathway; these are UvrA, UvrB, UvrC, and UvrD, all of which are well conserved in bacteria.NER deals with DNA-distorting lesions, in which an excinuclease removes a 12-to 13-nucleotide segment from a single strand centered around the lesion; the resulting gap is then filled in by repair synthesis [44].Loss of uvrB in H. pylori was shown to confer sensitivity to UV light, alkylating agents and low pH, suggesting that the H. pylori NER pathway is functional in repairing a diverse array of DNA lesions [45].H. pylori UvrD was shown to play a role in repairing DNA damage and limiting DNA recombination, indicating it functions to ultimately maintain genome integrity [46].The methyl-directed mismatch repair system (MMR), consisting of MutS1, MutH, and MutL, is conserved in many bacteria and eukaryotes, and it plays a major role in maintaining genetic stability.MMR can liberate up to 1000 nucleotides from one strand during its function to correct a single mismatch arising during DNA replication [47].Notably, MMR does not exist in H. pylori, contributing to the high mutation rates observed in H. pylori [17].H. pylori has a MutS homologue that belongs to the MutS2 family.H. pylori MutS2 was shown to bind to DNA structures mimicking recombination intermediates and to inhibit DNA strand exchange, thus it may play a role in maintaining genome integrity by suppressing homologous and homeologous DNA recombination [48].In addition, H. pylori MutS2 appears to play a role in repairing oxidative DNA damage, specifically 8-oxo-guanine [49].Damaged bases can be repaired by a variety of glycosylases that belong to the base excision repair (BER) pathway.All glycosylases can excise a damaged base resulting in an apurinic/apyrimidinic (AP) site, while some of them additionally nick the DNA deoxyribose-phosphate backbone (via an AP lyase activity).H. pylori harbors the glycosylase genes ung, mutY, nth, and magIII, whereas several other genes appear to be absent from the H. pylori genome, e.g.tag, alkA, and mutM.The H. pylori endonuclease III (nth gene product), which removes oxidized pyrimidine bases, was shown to be important in establishing longterm colonization in the host [50].The H. pylori MutY glycosylase is functional in removing adenine from 8-oxoG:A mispair, and the loss of MutY leads to attenuation of the colonization ability [51][52][53].To repair DNA double strand breaks and blocked replication forks, H. pylori is equipped with an efficient system of DNA recombinational repair, which is the main focus of this review (See section 4).

H. pylori response to DNA damage
Many bacteria encode a genetic program for a coordinated response to DNA damage called the SOS response.The best known E. coli SOS response is triggered when RecA binds ssDNA, activating its co-protease activity towards LexA, a transcriptional repressor [54].Cleavage of LexA results in transcriptional induction of genes involved in DNA repair, lowfidelity polymerases, and cell cycle control.However, the H. pylori genome contains neither a gene for LexA homolog nor the genes for low-fidelity polymerases, and an SOS response pathway seems to be absent in H. pylori [12,13].To define pathways for an H. pylori DNA damage response, Dorer et al. [55] used cDNA based microarrays to measure transcriptional changes in cells undergoing DNA damage.In both ciprofloxacin treated cells and the ΔaddA (a major DNA recombination gene, see section 4.4 below) mutant cells, the same set of genes were induced which include genes required for energy metabolism, membrane proteins, fatty acid biosynthesis, cell division, and some translation factors, although the contribution of these genes to survival in the face www.intechopen.com of DNA damage is not understood.No DNA repair genes, a hallmark of the SOS response, were induced in either the antibiotic-treated cells or the recombination gene deleted strain.Surprisingly, several genes involved in natural competence for DNA transformation (com T4SS components comB3, comB4 and comB9) were induced significantly.Indeed, natural transformation frequency was shown to be increased under DNA damage conditions.Another DNA damage-induced gene was a lysozyme-encoding gene.Experimental evidence was provided that a DNA damage-induced lysozyme may target susceptible cells in culture and provide a source of DNA for uptake [55].Taken together, DNA damage (mainly DSBs in their experiments) induces the capacity for taking up DNA segments from the neighboring cells of the same strain (homologous) or co-colonizing strain (homeologous) that may be used for recombinational DNA repair.

Mechanisms of DNA recombinational repair known in model bacteria
Although the bulk of DNA damage affects one strand of a duplex DNA segment, occasionally both DNA strands opposite each other are damaged; the latter situation necessitates recombinational repair using an intact homologous DNA sequence [56,57].DNA double-strand breaks (DSB) occur as a result of a variety of physical or chemical insults that modify the DNA (e.g.DNA strands cross-links).In addition, if a replication fork meets damaged bases that cannot be replicated, the fork can collapse leading to a DSB.In E. coli, 20-50% of replication forks require recombinational repair to overcome damage [58].Homologous recombinational repair requires a large number of proteins that act at various stages of the process [56].The first stage, pre-synapsis, is the generation of 3' singlestranded (ss) DNA ends that can then be used for annealing with the homologous sequence on the sister chromosome.In E. coli, the two types of two-strand lesions (double strand end and daughter strand gap) are repaired by two separate pathways, RecBCD and RecFOR, respectively [57].The second and most crucial step in DNA recombination is the introduction of the 3' DNA overhang into the homologous duplex of the sister chromosome, termed synapsis.This is performed by RecA in bacteria.RecA binds to ssDNA in an ATPdependent manner, and RecA-bound ssDNA (in a right-handed helix structure) can invade homologous duplex DNA and mediate strand annealing, accompanied by extrusion of the other strand that can pair with the remaining 5' overhang of the DSB (called D-loop formation).During DNA recombination, the single stranded DNA (ssDNA) is always coated (protected) by ssDNA-binding protein (SSB), which has a higher affinity to ssDNA than RecA.RecA needs to be loaded (during pre-synapsis stage), either by RecBCD or RecFOR, onto the generated ssDNA that is coated with SSB.During the third step in recombination, postsynapsis, RecA-promoted strand transfer produces a four-stranded exchange, or Holliday junctions (HJ) [59].The RecG and RuvAB helicases are two pathways that process the branch migration of HJ.Finally, RuvC resolves HJ in an orientation determined by RuvB, and the remaining nicks are sealed by DNA ligase.Several other genes (recJ, recQ, recN) are also required for recombination, although their functions are unclear [60,61].Single stranded exonuclease RecJ and RecQ helicase are sometimes needed to enlarge the gap for RecFOR to act [62].RecN, RecO, and RecF were found to be localized to distinct foci on the DNA in Bacillus subtilis cells after induction of DSBs [63].These proteins form active repair centers at DSBs and recruit RecA, initiating

DNA recombinational repair factors in H. pylori
While some genes that are predicted to be involved in DNA recombinational repair, including recA, recG, recJ, recR, recN, and ruvABC, were annotated from the published H. pylori genome sequences, many genes coding for the components that are involved in the pre-synapsis stage, such as RecBCD, RecF, RecO, and RecQ, were missing.Considering that H. pylori is highly genetic diverse with a high recombination frequency, this has been a big puzzle over the past decade.Recent studies revealed the existence of both pathways, AddAB (RecBCD-like) and RecRO, for initiation of DNA recombinational repair in H. pylori.In the following sections we will summarize the current understanding of DNA recombinational repair in H. pylori by reviewing the literature accumulated in recent years.

The central recombination protein RecA
The RecA protein is a central component of the homologous recombination machinery and of the SOS system in most bacteria.The relatively small RecA protein contains many functional domains including different DNA-binding sites and an ATP-binding site.E. coli RecA has also coprotease activities for the LexA repressor and other factors involved in SOS response.However, H. pylori genome does not contain a LexA homolog and an SOS response pathway is likewise absent in H. pylori.Thus, a coprotease activity may be dispensable for the H. pylori RecA protein.Nevertheless, RecA is required for DNA damage response observed in H. pylori, although the underlying mechanism is unclear [55].Before the genome era, the roles of H. pylori RecA in DNA recombination and repair have been studied genetically [65,66].H. pylori RecA (37.6 kDa protein) is highly similar to known bacterial RecA proteins.The H. pylori recA mutants were severely impaired in their ability to survive treatment with DNA damaging agents such as UV light, methyl methanesulfonate, ciprofloxacin, and metronidazole.H. pylori RecA also played a role in survival at low pH in a mechanism distinct from that mediated by urease [66].Disruption of recA in H. pylori abolished general homologous recombination [65].Interestingly, H. pylori RecA protein is subject to posttranslational modifications that result in a slight shift in its electrophoretic mobility [67].One putative mechanism for RecA modification is protein glycosylation.H. pylori RecA protein was shown to be membrane associated, but this association is not dependent on the posttranslational modification.The RecA modification is required for full activity of DNA repair [67].In recent years, the phenotypes of H. pylori recA mutants have been further characterized in comparison with other mutants.Among the mutants of DNA recombination and repair genes, recA mutants displayed the most severe phenotypes.For example, recA mutants were much more sensitive to UV or Gamma radiation than the recB or recO single mutants, and were similar to the recBO double mutant [68][69][70].The recA mutants completely lost the ability to undergo natural transformation [68][69][70].The intra-genomic recombination frequency of the recA mutant was also much lower than that of the recR or recB single mutants [68,71].Finally, the recA mutants completely lost the ability to colonize mouse stomachs [69].In competition experiments (mixed infection with wild type and mutant strains), recA mutant bacteria were never recovered, while some addA or addB mutant bacteria were recovered from mouse stomachs.

Post-synapsis proteins RuvABC and RecG
In addition to the synapsis protein RecA, the genes for post-synapsis proteins (RuvABC and RecG) are also well conserved among bacteria [72].Genes for RuvABC proteins are present in H. pylori, thus H. pylori seems to be able to restore Holliday Junctions in a similar way to E. coli.RuvC is a Holliday junction endonuclease that resolves recombinant joints into nicked duplex products.A ruvC mutant of H. pylori was more sensitive (compared to the wild type) to oxidative stress and other DNA damaging agents including UV light, mitomycin C, levofloxacin and metronidazole [73].As Macrophage cells are known to produce an oxidative burst to kill bacterial pathogens, the survival of H. pylori ruvC mutant within macrophages was shown to be 100-fold lower than that of the wild type strain [73].Furthermore, mouse model experiments revealed that the 50% infective dose of the ruvC mutant was approximately 100-fold higher than that of the wild-type strain.Although the ruvC mutant was able to establish colonization at early time points, infection was spontaneously cleared from the murine gastric mucosa over long periods (36 to 67 days) [73].This was the first experimental evidence that DNA recombination processes are important for establishing and maintaining long-term H. pylori infection.Further studies suggested that RuvC function and, by inference, recombination facilitate bacterial immune evasion by altering the adaptive immune response [74], although the underlying mechanisms remain obscure.RuvAB proteins are involved in the branch migration of Holliday junctions.The annotated H. pylori RuvB (HP1059) showed extensive homology (52% sequence identity) to E. coli RuvB, particularly within the helicase domains.However, unlike in E. coli, ruvA, ruvB, and ruvC are located in separate regions of the H. pylori chromosome, which may predict possible functional differences.In contrast to E. coli ruvB mutants, which have moderate susceptibility to DNA damage, the H. pylori ruvB mutant has intense susceptibility to UV, similar to that of a recA mutant [75].Similarly, the H. pylori ruvB mutant has a significantly diminished MIC (minimal inhibitory concentration) for ciprofloxacin, an agent that blocks DNA replication fork progression, to the same extent as the recA mutant.In agreement with these repair phenotypes, the ruvB mutant has almost completely lost the ability of natural transformation of exogenous DNA (frequency of <10 −8 ), similar to the recA mutant.In an assay measuring the intra-genomic recombination (deletion frequency between direct repeats), the ruvB mutants displayed significantly (four-to sevenfold) lower deletion frequencies than the background level.All four phenotypes of the ruvB mutant suggested that H. pylori RuvAB is the predominant pathway for branch migration in DNA recombinational repair [75].In E. coli, an alternative pathway processing branch migration of Holliday junctions is the RecG helicase.In marked contrast to E. coli, H. pylori recG mutants do not have defective DNA repair, as measured by UV-light sensitivity and ciprofloxacin susceptibility [76].Furthermore, H. pylori recG mutants have increased frequencies of intergenomic recombination and deletion, suggesting that branch migration and Holliday junction resolution are more efficient in the absence of RecG function [75,76].Thus, the effect of H. pylori RecG seems to be opposite to that of the RuvAB helicase.In the RuvABC pathway, the RuvC endonuclease nicks DNA, catalyzing Holliday junction resolution into doublestranded DNA.Although the resolvase in the RecG pathway has not been completely www.intechopen.comA Recombination Puzzle Solved: Role for New DNA Repair Systems in Helicobacter pylori Diversity/Persistence 9 elucidated, it has been hypothesized that RusA may serve this function in E. coli [77].By introducing E. coli rusA into H. pylori ruvB mutants, the wild-type phenotypes for DNA repair and recombination were restored [75].A hypothesis was proposed that RecG competes with RuvABC for DNA substrates but initiates an incomplete repair pathway (due to the absence of the RecG resolvase RusA) in H. pylori, interfering with the RuvABC repair pathway [75].

H. pylori RecN
Bacterial RecN is related to the SMC (structure maintenance of chromosome) family of proteins in eukaryotes, which are key players in a variety of chromosome dynamics, from chromosome condensation and cohesion to transcriptional repression and DNA repair [78].SMC family proteins have a structural characteristic of an extensive coiled-coil domain located between globular domains at the N-and C-termini that bring together Walker A and B motifs associated with ATP-binding [79].E. coli RecN is strongly induced during the SOS response and was shown to be involved in RecA-mediated recombinational repair of DSBs [64].In Bacillus subtilis, RecN was shown to be recruited to DSBs at an early time point during repair [63,80,81].In vitro, RecN was shown to bind and protect 3' ssDNA ends in the presence of ATP [82].
In the published H. pylori genome sequence [12], HP1393 was annotated as a recN gene homolog.The H. pylori recN mutant is much more sensitive to mitomycin C, an agent that predominantly causes DNA DSBs, indicating RecN plays an important role in DSB repair in H. pylori [83].In normal laboratory growth conditions, an H. pylori recN mutant does not show a growth defect, but its survival is greatly reduced under oxidative stress which resembles the in vivo stress condition.While very little fragmented DNA was observed in either wild type or recN mutant strain when cells were cultured under normal microaerobic conditions; after oxidative stress treatment the recN mutant cells had a significantly higher proportion of the DNA as fragmented DNA than did the wild type [83].Similar roles of RecN in protection against oxidative damage have been demonstrated in Neisseria gonorrhoeae [84,85].In addition, the H. pylori recN mutant is much more sensitive to low pH than the wild type strain, suggesting that RecN is also involved in repair of acid-induced DNA damage [83].This could be relevant to its physiological condition, as H. pylori appears to colonize an acidic niche on the gastric surface [41].As mentioned in the sections above, loss of H. pylori RecA, RuvB or RuvC functions results in a great decrease of DNA recombination frequency.Similarly, the H. pylori recN mutant has a significant decrease of DNA recombination frequency, suggesting that RecN is a critical factor in DNA recombinational repair [83].In contrast, loss of UvrD or MutS2 in H. pylori resulted in an increase of DNA recombination frequency [46,48].Suppression of DNA recombination by UvrD or MutS2, and facilitation of DNA recombination by RecN, may play a role in coordinating DNA repair pathways.Recombinational repair could be mutagenic due to homeologous recombination or cause rearrangement due to recombination with direct repeat sequences.In addition, recombinational repair systems are much more complex and require more energy to operate, compared to nucleotide excision repair (NER) and base excision repair (BER) systems.Thus UvrD, as a component of NER, and MutS2 as a likely component of a BER (8-oxoG glycosylase) system [49], both suppress DNA recombination.Both NER and BER systems would be expected to continuously function in low stress conditions.Under a severe stress condition when large amounts of DSBs are formed, RecN perhaps recognizes DSBs and recruits proteins required for initiation of DNA recombination.The role of H. pylori RecN in vivo has been demonstrated, as the recN-disrupted H. pylori cells are less able to colonize hosts than wild type cells [83].However, the mouse colonization phenotype of the recN strain seems to be less severe than those observed for the recA or ruvC mutants.In contrast to RecA or RuvC which are major components of DNA recombination machinery, RecN is a protein specific for repairing DSBs by linking DSB recognition and DNA recombination initiation.It was proposed that the attenuated ability to colonize mouse stomachs by recN cells was mainly due to the strain's failure to repair DSBs through a DNA recombinational repair pathway.

AddAB helicase-nuclease
DNA helicases play key roles in many cellular processes by promoting unwinding of the DNA double helix [86].Bacterial genomes encode a set of helicases of the DExx family that fulfill several, sometimes overlapping functions.Based on the sequence homology, bacterial RecB, UvrD, Rep, and PcrA were classified as superfamily I (SF1) helicases [86][87][88].In the well-studied E. coli, RecBCD form a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break.RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site).The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA [89].Another bacterial enzyme complex AddAB, extensively studied in Bacillus subtilis, has both nuclease and helicase activities similar to those of RecBCD enzyme [90,91].The genes for RecBCD or AddAB were missing in the published H. pylori genome [12,13].However, HP1553 from strain 26695 was annotated as a gene encoding a putative helicase [12], and the corresponding gene from strain J99 was annotated as pcrA [13].Amino acid sequence alignment of HP1553 to E. coli RecB (or to B. subtilis AddA) revealed 24% identity (to both heterologous systems) at the N-terminal half (helicase domain), and no significant homology at the C-terminal half (including nuclease domain).Thus, HP1553 could be a RecB (or AddA)-like helicase [69,92].Furthermore, by using the highly conserved AddB nuclease motif "GRIDRID" in BLAST search, HP1089 was identified as the putative AddB homolog [69].Now it is accepted that HP1553 and HP1089 are termed addA and addB respectively in H. pylori with a reminder that previous recB [20,68,70,92] was the equivalent of addA [69,71,93].Both genes addA and addB are present in 56 H. pylori clinical isolates from around the world [94]; thus they are considered core genes that are not strain variable.The biochemical activities of H. pylori AddAB helicase-nuclease have been demonstrated [69].Cytosolic extracts from wild-type H. pylori showed detectable ATP-dependent nuclease activity with ds DNA substrate, while the addA and addB mutants lack this activity.Cloned H. pylori addA and addB genes express ATP-dependent exonuclease in E. coli cells.These genes also conferred ATP-dependent DNA unwinding (helicase) activity to an E. coli recBCD deletion mutant, indicating that they are the structural genes for this enzyme [69].The roles of individual (helicase, exonuclease) activity of the AddA and AddB in DNA repair, recombination, and mouse infection have been further studied by site-directed mutagenesis approach [93].H. pylori addA and addB mutant strains showed heightened sensitivity to mitomycin C and the DNA gyrase inhibitor ciprofloxacin, both of which lead to DNA ds breaks [69,92].The

www.intechopen.com
A Recombination Puzzle Solved: Role for New DNA Repair Systems in Helicobacter pylori Diversity/Persistence 11 level of sensitivity was similar to that seen for a recA mutant, but more severe than for the recN mutant.It is thus concluded that AddAB plays a major role in the repair of DNA ds breaks [69,92].On the other hand, the addA and addB mutants were markedly less sensitive to UV irradiation than a recA mutant, suggesting that AddAB does not play a major role in repair of UV damage in H. pylori [69].AddA was shown to be important for H. pylori protection against oxidative stress-induced damage, as the addA mutant cells were significantly more sensitive to oxidative stress and contained a large amount of fragmented DNA [92].Furthermore, loss of AddA resulted in reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins (babA to babB) [69].Finally, it was shown that the addA and addB mutant strains display a significantly attenuated ability to colonize mouse stomachs, in both competition experiments and during single-strain infections [69,92].While addA and addB are adjacent in the chromosome in most bacteria, including other epsilon Proteobacteria, this is not the case in H. pylori.However, the phenotypes of H. pylori addA and addB mutants are indistinguishable.Thus, it was proposed [69] that the AddA and AddB act together in a complex, as do the RecBCD polypeptides and AddAB polypeptides of other bacteria.If so, the control of the unlinked H. pylori addA and addB genes to maintain the proper stoichiometry of the two polypeptides remains an interesting question.Regarding the role of H. pylori AddA in DNA recombination during natural transformation, conflicting results were reported from different studies.The addA (note: it was named recB in certain references) mutant showed enhanced [68,70], decreased [20,71,92], or no change [27,69] in transformation frequency.Indeed, a high degree of variability (>100-fold) in transformation frequency in H. pylori was observed between different strains and different experiments.The use of different assay systems may partly explain the discrepancy in transformation results.For example, the total genomic DNA from antibiotic-resistant strain was used for the transformation assay in certain studies, while in others the defined linear DNA fragments of small size [92].Use of the transformation frequency as an indicator of DNA recombination frequency is based on the assumption that the wild type H. pylori and its isogenic rec strains are equally competent for DNA uptake.However, it is now known that this assumption is not valid because DNA damage triggers genetic exchange in H. pylori [55].H. pylori addA mutant cells suffered more DNA damage [92], and have an enhanced competence for DNA uptake [55].Thus, the accumulation of unrepaired DNA damage and subsequent poor growth, as well as unknown strain differences, could be the main cause of the high degree of variability in H. pylori transformation frequency [27].

H. pylori RecRO pathway
RecFOR is a highly conserved DNA recombination pathway in bacteria, and is mainly used for ssDNA gap repair [72].In the published H. pylori genome sequences, only the recR gene was annotated [12,13].Although RecF historically served as a reference for RecFOR pathway, it is absent from genomes of many bacteria including H. pylori [72].By bioinformatics analysis, Marsin et al [68] identified HP0951 as a novel RecO orthologue, although its sequence identity with the E. coli protein is lower than 15%.Recent studies in E. coli indicated that RecOR in the absence of RecF can perform recombination by loading RecA [95,96].Whereas the RecO protein can displace ssDNA-binding protein (SSB) and www.intechopen.combind to ssDNA, RecR is the key component for loading RecA onto ssDNA [95,97].Likely, the RecRO pathway (with no RecF) is present in H. pylori.The recR and recO mutants showed marked sensitivity to DNA damaging agents metronidazole and UV light, indicating roles of RecR and RecO in DNA repair.Unlike the addA (recB) mutant, the recR and recO mutants did not show significant sensitivity to ionizing radiation (IR) and to mitomycin C [68,71], suggesting that RecRO pathway is not responsible for repairing DNA damage induced by these agents, most likely double strand breaks.This is in contrast to E. coli where the RecFOR pathway sometimes substitutes for the RecBCD pathway and in Deinococcus radiodurance where the RecFOR pathway plays a major role in double strand break repair [98,99].On the other hand, H. pylori recR and recO mutants were shown to be much more sensitive to oxidative stress and to acid stress than the wild type strain [71], indicating that H. pylori RecRO pathway is involved in repairing DNA damage induced by these stress conditions.The addA recO double mutant (deficient in both AddAB and RecRO pathways) was significantly more sensitive to atmospheric oxygen than the recO single mutant, indicating that both RecRO and AddAB pathways are important for survival of oxidative damage.Similar roles of the RecBCD and the RecFOR pathways for survival of oxidative damage were also observed in E. coli [57,100] and in Neisseria gonorrhoeae [84].In those bacteria, however, the RecBCD appeared to be the predominant (over the RecFOR) repair pathway for oxidative damage.Our results suggest that the two pathways in H. pylori play similarly important roles in repairing oxidative stress-derived DNA damage [71].In accordance with the sensitivity to oxidative and acid stress in vitro, H. pylori recR and recO mutants were shown to be less able to colonize mouse stomachs [71].Furthermore, the mouse colonization ability of the addA recO double mutant was significantly lower than that of the addA or recO single mutant.Therefore, both AddAB-and RecRO-mediated DNA recombinational repair in H. pylori play an important role in bacterial survival and persistent colonization in the host.Although differing results regarding the effect of addA gene on transformation frequency were reported by different research groups, it was agreed that the RecRO-pathway is not involved in recombination of exogenous DNA into the H. pylori genome in the process of transformation [68,71].The RecRO pathway is known to have a major role in intragenomic recombination at repeat sequences [101].Using an assay to assess the deletion frequency resulting from recombination on direct repeat sequences (358 bp long), Marsin et al [68] showed that the recR and recO mutants exhibited a statistically significantly lower deletion frequency than the wild type strain, suggesting a role of RecRO in intragenomic recombination.Recently we adopted a similar assay using DNA constructs (deletion cassettes) that contain identical repeat sequences of different length (IDS100 and IDS350) [71].The results indicated that the intra-genomic recombination of 100 bp-long direct repeat sequences in H. pylori is partially dependent on RecR and RecA, yet a large portion of the recombination event is RecA-independent.This is basically in agreement (with small variance) with the results of Aras et al [35] who reported that the repeat sequences of 100 bp or shorter recombined through a RecA-independent pathway.For the deletion cassette containing repeat sequences of 350 bp in length, inactivation of recR or recA resulted in a significant 4-fold or 35-fold decrease respectively in deletion frequency, indicating that RecR plays a significant role in recombination of IDS350, while this recombination was highly dependent on RecA.

Concluding remarks and perspectives
Severe Helicobacter pylori-mediated gastric diseases are associated with the bacterium's persistence in the host and its adaptability to host differences, which in turn is associated with its remarkable genetic variability.DNA recombination is an extraordinarily frequent event in H. pylori, and this manifests itself into a bacterium with unusual flexibility in stresscombating enzymes, repair mechanisms, and other adaptability characteristics.Nearly every H. pylori recombination-related gene studied thus far by a gene directed mutant analysis approach has documented they are individually important in stomach colonization ability; this underscores the importance of these recombination repair processes in bacterial survival in the host.It is well recognized that homologous DNA recombination is a special system in bacteria for repairing stalled replication forks and double strand breaks, while generating genetic diversity as an advantageous byproduct [102].H. pylori may be an especially fruitful organism in which to learn the ultimate boundaries in roles of recombination repair enzymes, as H. pylori is subject to intense and prolonged host mediated stress and it displays an enormous genetic diversity.Substantial progress has been made recently in unraveling the complex systems of DNA recombinational repair in H. pylori.As expected, whole genome sequencing has been a powerful tool to aid in identifying recombination-related proteins in H. pylori.For example, recA, recR, recN, and ruvABC were identified and confirmed to play important roles in H. pylori as could be expected from results for other bacteria.Some recombination-related proteins (e.g.MutS2, RecG), however, play unique roles in H. pylori.Most of the genes for the major components of the two pre-synapsis pathways (RecBCD and RecFOR) were not annotated from H. pylori genome sequences, which drove researchers' interest to search for additional novel systems required for H. pylori DNA recombinational repair.Recent studies revealed the existence of both pathways, AddAB and RecRO, in H. pylori.Although they display a limited level of sequence homology to the known recombination enzymes, both AddAB and RecRO were shown to play important roles in H. pylori DNA recombinational repair, conferring resistance to oxidative and acid stress.The major components of DNA recombinational repair machinery in H. pylori are listed in Table 1.H. pylori RecN protein may recognize DNA double strand breaks and recruits AddAB helicase-nuclease complex for further processing.While not being involved in repair of DNA double strand breaks, H. pylori RecRO proteins play a major role in intra-genomic recombination at repeat sequences.Both pre-synapsis pathways (AddAB and RecRO) require RecA for catalyzing DNA strand exchange (synapsis) and H. pylori RuvABC is the predominant pathway for DNA branch migration and Holliday Junction resolution (postsynapsis).Although the major functions of these components are similar to those observed in model bacteria, some novel attributes of these components have been discovered, which may be related to the highly-specific lifestyle of H. pylori.Additional new components that work synergistically with these pathways could be found in this unique bacterium via future biochemical and genetic approaches.
Recombination Puzzle Solved: Role for New DNA Repair Systems in Helicobacter pylori Diversity/Persistence 13 www.intechopen.comA

Table 1 .
[12]refers to the gene number in the genome sequence of strain 26695[12].(b) DSB (double strand breaks) damage refers to those damages caused e.g. by ionizing radiation, mitomycin C, or ciprofloxacin.H. pylori genes involved in DNA recombinational repair 7. References