Critical Role of RecN in Recombinational DNA Repair and Survival of Helicobacter pylori

Abstract

Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more sensitive than its parent to mitomycin C, an agent predominantly causing DNA double-strand breaks. The recN strain was unable to survive exposure to either air or acid as well as the parent strain, and air exposure resulted in no viable recN cells recovered after 8 h. In oxidative stress conditions (i.e., air exposure), a recN strain accumulated significantly more damaged (multiply fragmented) DNA than the parent strain. To assess the DNA recombination abilities of strains, their transformation abilities were compared by separately monitoring transformation using H. pylori DNA fragments containing either a site-specific mutation (conferring rifampin resistance) or a large insertion (kanamycin resistance cassette). The transformation frequencies using the two types of DNA donor were 10-and 50-fold lower, respectively, for the recN strain than for the wild type, indicating that RecN plays an important role in facilitating DNA recombination. In two separate mouse colonization experiments, the recN strain colonized most of the stomachs, but the average number of recovered cells was 10-fold less for the mutant than for the parent strain (a statistically significant difference). Complementation of the recN strain by chromosomal insertion of a functional recN gene restored both the recombination frequency and mouse colonization ability to the wild-type levels. Thus, H. pylori RecN, as a component of DNA recombinational repair, plays a significant role in H. pylori survival in vivo.

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

“…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].…”

“…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].…”

“…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.…”

“…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.…”