Biology subjects

Muniyappa, K.

Publications and source records attributed to Muniyappa, K..

3 recordsLinked to original sources

bioRxiv2024

Cyclic di-AMP regulates genome stability and drug resistance in Mycobacterium through RecA-dependent and -independent recombination

In Escherichia coli, RecA plays a central role in the rescue of stalled replication forks, double-strand break (DSB) repair, homologous recombination (HR) and induction of the SOS response. While the RecA-dependent pathway is dominant, alternative HR pathways that function independently of RecA do exist, but relatively little is known about the underlying mechanism. Several studies have documented that a variety of proteins act either as positive or negative regulators of RecA to ensure high-fidelity HR and genomic stability. Along these lines, we previously demonstrated that the second messenger cyclic di-AMP binds to mycobacterial RecA proteins, but not E. coli RecA, and inhibits its DNA strand exchange activity in vitro via the disassembly of RecA nucleoprotein filaments. Herein, we demonstrate that Mycobacterium smegmatis {Delta}disA cells, which lack c-di-AMP, exhibit increased DNA recombination, higher frequency of mutation and gene duplications during RecA-dependent and RecA-independent DSB repair. We also found that c-di-AMP regulates SOS response by inhibiting RecA-mediated self-cleavage of LexA repressor and its absence enhances drug resistance in M. smegmatis {Delta}disA cells. Together, our results uncover a role of c-di-AMP in the maintenance of genomic stability through modulation of DSB repair in M. smegmatis. SignificanceCyclic di-AMP is a second messenger present in bacteria and archaea and is implicated in a variety of functions in the cell, including DNA repair, cell wall metabolism, virulence, and gene expression. We show here that it maintains genome stability in Mycobacterium by regulating RecA-dependent and -independent DNA recombination pathways. It also regulates SOS response by inhibiting the self-cleavage of LexA by mycobacterial RecA. Absence of c-di-AMP leads to higher drug resistance in Mycobacterium.

molecular biology↗

bioRxiv2024

Saccharomyces cerevisiae Rev7 regulates DSB repair pathway choice through binding and blocking Mre11 nuclease and Rad50 ATPase activities

Recent studies have shown that, in human cancer cells, the tetrameric Shieldin complex (comprising REV7, SHLD1, SHLD2, and SHLD3) facilitates non-homologous end-joining (NHEJ) while blocking homologous recombination (HR). Surprisingly, several eukaryotic species lack SHLD1, SHLD2 and SHLD3 orthologs, suggesting that Rev7 may leverage an alternative mechanism to regulate the double-strand break (DSB) repair pathway choice. Exploring this hypothesis, we discovered that Saccharomyces cerevisiae Rev7 physically interacts with the Mre11-Rad50-Xrs2 (MRX) subunits, impedes G-quadruplex DNA synergised-HU-induced toxicity and facilitates NHEJ, while antagonizing HR. Notably, we reveal that a 42-amino acid C-terminal fragment of Rev7 binds to the subunits of MRX complex, protects rev7{Delta} cells from G-quadruplex DNA-HU-induced toxicity, and promotes NHEJ by blocking HR. By comparison, the N-terminal HORMA domain, a conserved protein-protein interaction module, was dispensable. We further show that the full-length Rev7 impedes Mre11 nuclease and Rad50s ATPase activities without affecting the latters ATP-binding ability. Combined, these results provide unanticipated insights into the functional interaction between the MRX subunits and Rev7 and highlight a mechanism by which Rev7 facilitates DSB repair via NHEJ, and attenuation of HR, by blocking Mre11 nuclease and Rad50s ATPase activities in S. cerevisiae. IMPACT STATEMENTThe mechanisms steering DNA double-strand break repair pathway choice is a topic of intense investigation, but remains incompletely understood. Our findings suggest that yeast Rev7 promotes DSB repair via NHEJ and inhibits homologous recombination by blocking Mre11 nuclease and Rad50s ATPase activities.

molecular biology↗

bioRxiv2022

Dual targeting of Saccharomyces cerevisiae Pso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks

Repair of DNA interstrand crosslinks (ICLs) involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that ICL- inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA (mtDNA), and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mtDNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we assessed the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains one mitochondrial targeting sequence (MTS) and two nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the MTS. Further analysis revealed that Pso2 is a dual-localized ICL repair protein; it can be imported into both nucleus and mitochondria, and that genotoxic agents enhance its abundance in the latter. While MTS is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the two NLS motifs are functionally redundant. Ablation of MTS abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both NLS motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between MTS and NLS motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual-targeting of Pso2 into two DNA-bearing organelles is discussed.

genetics↗