Post-doctoral research fellow, University of Edinburgh and University of Ghana
Development of novel antibiotics targeting the DNA double-strand break repair pathway
The current antibiotics in use at clinics are no longer effective against the treatment of infections caused by pathogenic bacteria. This dreadful phenomenon is caused by the continuous exposure of pathogenic bacteria to the available antibiotics, which has led to an inherent modification of the bacteria, thereby allowing it to survive in the presence of the antibiotics. Consequently, it has become imperative to discover new effective antibiotics, as well as understand the various mechanisms that are used by these infectious bacteria for survival in the presence of the antibiotics.
My current research is focused on investigating the repair of DNA double-strand breaks (DSBs) as a suitable cellular target in bacteria for the development of new antibiotics. DSB is a form of DNA damage that must be repaired to ensure cell survival. Even though DSBs can occur in both bacteria and humans, the proteins that are used for repair are different. This phenomenon presents a unique opportunity for inhibiting the repair of DSBs in pathogenic bacteria without affecting the same repair event in humans.
The lab of Dr Patrick K. Arthur (University of Ghana) has isolated a wide collection of crude (impure) extracts from fungal sources. During the initial phase of this research, all the crude extracts were screened against selected pathogenic bacteria to identify potential candidates that were capable of either generating DSBs or inhibiting the repair pathway. Afterwards, pairs of these potential extracts that synergistically generated DSBs and inhibited the repair pathway were selected. We are currently isolating the active compound in each of the selected pairs of crude extracts. These active compounds, in the purified state, would be tested against the relevant pathogenic bacteria to ascertain their potential as novel drugs that target the DSB repair pathway. This research highlights the progress that we have made in the quest to develop novel antibiotics against pathogenic and resistant bacterial strains.
Abstract: Long DNA palindromes are sites of genome instability (deletions, amplification, and translocations) in both prokaryotic and eukaryotic cells. In Escherichia coli, genetic evidence has suggested that they are sites of DNA cleavage by the SbcCD complex that can be repaired by homologous recombination. Here we obtain in vivo physical evidence of an SbcCD-induced DNA double-strand break (DSB) at a palindromic sequence in the E. coli chromosome and show that both ends of the break stimulate recombination. Cleavage is dependent on DNA replication, but the observation of two ends at the break argues that cleavage does not occur at the replication fork. Genetic analysis shows repair of the break requires the RecBCD recombination pathway and PriA, suggesting a mechanism of bacterial DNA DSB repair involving the establishment of replication forks.
Pub.: 18 Mar '08, Pinned: 25 Jan '18
Abstract: Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways.
Pub.: 12 Jan '11, Pinned: 25 Jan '18
Abstract: DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.
Pub.: 24 Dec '13, Pinned: 25 Jan '18
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