Biology subjects

Simmons, L. A.

Publications and source records attributed to Simmons, L. A..

4 recordsLinked to original sources

bioRxiv2018

A bacterial DNA repair pathway specific to a natural antibiotic

All organisms possess several DNA repair pathways to maintain the integrity of their genetic material. Although there are several DNA repair pathways that are well understood, we recently identified several genes in Bacillus subtilis that are important for surviving treatment with drugs that damage DNA. Here, we report a drug specific DNA repair pathway in B. subtilis. We identified genes coding for a previously uncharacterized helicase and exonuclease, mrfA and mrfB, respectively. Deletion of mrfA and mrfB resulted in sensitivity to the DNA damaging agent mitomycin C, but not other types of DNA damage. We found that MrfAB operate independently of canonical nucleotide excision repair, forming a novel excision repair pathway in bacteria. A phylogenetic analysis demonstrates that MrfAB homologs are present in diverse bacterial phyla, and a cross-complementation assay shows that MrfAB function is conserved in closely related species. Mitomycin C is a natural antibiotic that is produced by the soil dwelling bacterium Streptomyces lavendulae, and B. subtilis is also a soil dwelling organism. The specificity of the {Delta}mrfAB phenotype suggests that MrfAB have been adapted as a countermeasure to mitomycin producing bacteria.\n\nAbbreviated SummaryBacteria possess DNA repair pathways to maintain the integrity of their genetic material. The helicase MrfA and the exonuclease MrfB are part of a mitomycin C specific DNA repair pathway in Bacillus subtilis. Despite being present in many bacterial species, MrfAB activity in repairing MMC damaged DNA appears to be restricted to closely related species, suggesting that these proteins have likely been adapted to the specific needs of each bacterium.

microbiology↗

bioRxiv2018

DdcA antagonizes a bacterial DNA damage checkpoint

Bacteria coordinate DNA replication and cell division, ensuring that a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage or impediments to DNA replication, a cell cycle checkpoint is activated to delay cell division by expressing a cell division inhibitor. The prevailing model for bacterial DNA damage checkpoints is that activation of the DNA damage response and protease mediated degradation of the cell division inhibitor is sufficient to regulate the checkpoint process. Our recent genome-wide screens identified the gene ddcA as critical for surviving exposure to a broad spectrum of DNA damage. The ddcA deletion phenotypes are dependent on the checkpoint enforcement protein YneA. We found that expression of the checkpoint recovery proteases could not compensate for ddcA deletion. Similarly, expression if ddcA could not compensate for the absence of the checkpoint recovery proteases, indicating that DdcA function is distinct from the checkpoint recovery step. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels or stability, further supporting the conclusion that DdcA regulates YneA through a proteolysis independent mechanism. Using a functional GFP-YneA we found that DdcA inhibits YneA activity independent of YneA localization, suggesting that DdcA may regulate YneA access to its target. These results uncover a regulatory step that is important for controlling the DNA damage checkpoint in bacteria, and suggests that the typical mechanism of degrading the checkpoint enforcement protein is insufficient to control the rate of cell division in response to DNA damage.\n\nAuthor SummaryAll cells coordinate DNA replication and cell division. When cells encounter DNA damage, the process of DNA replication is slowed and the cell must also delay cell division. In bacteria, the process has long been thought to occur using two principle modes of regulation. The first, is RecA coated ssDNA transmits the signal of DNA damage through inactivation of the repressor of the DNA damage (SOS) response regulon, which results in expression of a cell division inhibitor establishing the checkpoint. The second principle step is protease mediated degradation of the cell division inhibitor relieving the checkpoint. Recent work by our lab and others has suggested that this process may be more complex than originally thought. Here, we investigated a gene of unknown function that we previously identified as important for survival when the bacterium Bacillus subtilis is exposed to DNA damage. We found that this gene negatively regulates the cell division inhibitor, but is functionally distinct from the checkpoint recovery process. We provide evidence that this gene functions as an antagonist to establishing the DNA damage checkpoint. Our study uncovers a novel layer of regulation in the bacterial DNA damage checkpoint process challenging the longstanding models established in the bacterial DNA damage response field.

microbiology↗

bioRxiv2018

Cryptic adaptor protein interactions regulate DNA replication initiation

DNA replication is a fundamental biological process that is tightly regulated in all living cells. In bacteria, the master regulator DnaA controls when and where replication begins by building a step-wise complex that loads the replicative helicase onto chromosomal DNA. In many bacteria, DnaA requires the adaptor proteins DnaD and DnaB to aid DnaA during helicase loading. How DnaA, its adaptors, and the helicase form a complex at the origin is largely unknown. In this study, we addressed this long-standing question by disassembling the initiation proteins into their individual domains and testing all possible pair-wise combinations in a bacterial two-hybrid assay. Here we report a full description of the cryptic interaction sites used by the helicase loading machinery from Bacillus subtilis. In addition, we investigated how complex formation of the helicase loading machinery is regulated by the checkpoint protein SirA, which is a potent replication inhibitor in sporulating cells. We found that SirA and the DnaD adaptor bind overlapping sites on DnaA, and therefore SirA acts as a competitive inhibitor to block initiation. The interaction between DnaA and DnaD was also mapped to the same DnaA surface in the human pathogen Staphylococcus aureus, demonstrating the broad conservation of this interface. Therefore, our approach has unveiled key protein interactions essential for initiation and is widely applicable for mapping interactions in other signaling pathways that are governed by cryptic binding surfaces.\n\nAuthor SummaryIn order to proliferate, bacteria must first build a step-wise protein complex on their chromosomes that determines when and where DNA replication begins. This protein complex is assembled through dynamic interactions that have been difficult to study and remain largely uncharacterized. Here we show that by deconstructing the proteins into their constituent domains, the interactions used to build the initiation complex can be readily detected and mapped to single amino acid resolution. Using this approach, we demonstrate that DNA replication is controlled through conformational changes that dictate the availability of interaction surfaces. In addition, negative regulators can also block DNA replication by influencing complex formation so that cells survive inhospitable conditions. Initiation proteins from the model organism B. subtilis and the human pathogen S. aureus were both used to underscore the general applicability of the results to different bacterial systems. Furthermore, our general strategy for mapping dynamic protein interactions is suitable for many different signaling pathways that are controlled through cryptic interaction surfaces.

microbiology↗

bioRxiv2018

Discovery of a two protease DNA damage checkpoint recovery mechanism

The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells sense successful DNA repair and promote checkpoint recovery is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.

molecular biology↗