**4.1 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 [68-70]. The *recA* mutants completely lost the ability to undergo natural transformation [68-70]. 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

A Recombination Puzzle Solved: Role for New

pathway [75].

**4.3** *H. pylori* **RecN** 

the presence of ATP [82].

to colonize an acidic niche on the gastric surface [41].

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

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

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

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

strains), *recA* mutant bacteria were never recovered, while some *addA* or *addB* mutant bacteria were recovered from mouse stomachs.

#### **4.2 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 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].
