Biology subjects

Simmons, L.

Publications and source records attributed to Simmons, L..

5 recordsLinked to original sources

bioRxiv2026

DnaE uses strand displacement synthesis during Okazaki fragment repair

RNA serves the essential role of priming DNA replication for all organisms. In bacteria, it is estimated that more than 20,000 ribonucleotides accumulate on the lagging strand per round of replication. Despite the importance of RNA primers in initiating DNA synthesis, unresolved primers result in strand breaks and induction of the DNA damage response, leading to genome instability. In bacteria, the prevailing model suggests that DNA polymerase I (Pol I) replaces RNA primers with DNA during lagging strand replication. However, in Bacillus subtilis, we show that {Delta}polA cells have a near wild-type phenotype, demonstrating that cells lacking Pol I still perform efficient Okazaki fragment repair. To determine how cells compensate for the loss of Pol I, we tested the ability of two major replicative polymerases, DnaE and PolC, to participate in lagging strand replication in vitro. We found that DnaE replicates an Okazaki fragment as efficiently as Pol I using strand displacement synthesis. In contrast, PolC is unable to catalyze strand displacement synthesis, but can facilitate repair when combined with a nuclease or a gapped substrate. Together, our work shows that B. subtilis can use several different mechanisms to replicate the lagging strand, ensuring fidelity within the replication cycle.

microbiology↗

bioRxiv2025

DNA polymerase I is an efficient reverse transcriptase that mediates RNA-templated DNA repair synthesis

It is well established that during normal growth and under stress conditions ribonucleotides become nested in genomic DNA. Given the high frequency with which RNA is found in genomic DNA, it is unknown which DNA polymerase is responsible for catalyzing RNA-dependent DNA synthesis. In this work we establish bacterial DNA polymerase I (Pol I) as a robust reverse transcriptase. We tested several bacterial Pol I enzymes and found that all possess RT activity with B. subtilis Pol I showing RNA-dependent DNA synthesis activity similar to the Moloney Murine Leukemia Virus reverse transcriptase. We tested RNA embedded in DNA and found that replicative DNA polymerase PolC arrests at a single template ribonucleotide while, in contrast, Pol I is able to efficiently traverse long stretches of templated ribonucleotides. We conclude that Pol I is responsible for RNA templated DNA replication, allowing for replication forks to navigate RNA that persists in genomic DNA.

biochemistry↗

bioRxiv2025

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R-loops during transcription. R-loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis: DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photactivated localization microscopy. In this work, we find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo. SignificanceRNA-DNA hybrids (RDHs) are essential, transient intermediates in DNA replication, yet their presence significantly increases the susceptibility of the genome to damage. We characterized the single-molecule behavior of three proteins important for processing RDHs in Okazaki fragments in living bacteria. We find that enzyme activity is modulated by access to the replisome and nucleoid. Specifically, we find that DNA polymerase I is preferentially localized to the replisome, while FenA and RNase HIII dwell times at the replisome are very short and below our detection limit. Our work shows that Pol I, FenA, and RNase HIII turn over rapidly in cells, providing new insight into how lagging-strand replication is coordinated in vivo.

biophysics↗

bioRxiv2020

A GT-seq panel for walleye (Sander vitreus) provides a generalized workflow for efficient development and implementation of amplicon panels in non-model organisms.

Targeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals, but studies describing detailed workflows of GTseq panel development are rare. Here, we develop a dual-purpose GT-seq panel for walleye (Sander vitreus) and discuss trade-offs associated with different development and genotyping approaches. Our GT-seq panel was developed using restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We then conducted simulations to test the utility of loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We conducted three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. Optimization focused on reducing variation in amplification rate among loci and minimizing the proportion of off-target sequence, both of which are important considerations for developing large GT-seq panels. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (1) inexpensive Chelex extractions performed well for genotyping, (2) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was likely unnecessary, and (3) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings should aid in their construction in myriad taxa.

genomics↗

bioRxiv2018

Two essential replicative DNA polymerases exchange dynamically during DNA replication and after replication fork arrest

The replisome is the multi-protein complex responsible for faithful replication of chromosomal DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in live Bacillus subtilis cells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following both damage-independent and damage-dependent replication fork stress. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replisomal proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process, and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results show that DnaE provides a limited contribution to chromosomal replication and repair.

biophysics↗