Identification of pre-synaptic processing proteins from bacteroides fragilis

Abstract

The repair of DNA double-strand breaks (DSBs) is required for the survival of all organisms. In bacteria, DNA DSBs can occur during normal housekeeping processes such as DNA replication or by exogenous damage due to chemicals or radiation. DSBs will compromise the integrity of the genome if left un-repaired, and can be fatal to an organism. Repair of DSBs by homologous recombination (HR) replicates missing chromosomal regions before joining of the separated DNA ends. In Escherichia coli the HR repair steps are; pre-synapsis, synapsis and post-synapsis. In the pre-synaptic stage a DSB is processed into a 3′ single-strand overhang, the substrate required for strand invasion in the synapsis stage and the eventual repair of the DSB. At present there are three identified pre-synapsis systems involved in recombination in bacteria; represented by the AdnAB, AddAB and the RecBCD protein complexes. Each system functions in a similar manner but differ in the physical composition of the machinery. This project investigated the pre-synaptic system of Bacteroides fragilis NCTC9343. Genes encoding putative pre-synapsis proteins were initially identified through analysis of the NCTC9343 genome. The function of these proteins was investigated in vivo by rescue of a repair-deficient strain of E. coli. This demonstrated that Bacteroides fragilis encodes a two component system, where both genes products are required to work in concert for pre-synaptic processing of DSBs. The identified genes were BF2192 and BF2191, and have been renamed addA and addB, respectively. To further examine the role of the AddAB proteins in DSB repair, a Bacteroides fragilis strain with a deletion of addAB was constructed and shown to be extremely sensitive to DNA damaging agents. The AddAB complex was purified and found to be an ATP-dependant helicase and exonuclease that acted on double-stranded DNA ends. In conclusion, this project has identified the proteins involved in pre-synaptic processing of DSBs in B. fragilis NCTC9343, consisting of AddAB homologues, and shown their protective role in repair of DNA damage.