**5. Concluding remarks and perspectives**

12 DNA Repair

bind to ssDNA, RecR is the key component for loading RecA onto ssDNA [95, 97]. Likely,

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

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

the RecRO pathway (with no RecF) is present in *H. pylori*.

the host.

dependent on RecA.

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.
