Biology subjects

Lowder, F. C.

Publications and source records attributed to Lowder, F. C..

4 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↗

bioRxiv2022

Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids

Current models for Okazaki fragment maturation in eubacteria invoke RNA cleavage by RNase H, followed by strand displacement synthesis and 5' RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5'-3' flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on 5' flapped and nicked RNA-DNA hybrid substrates. We found that the 5' nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5' flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that {Delta}polA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5' nuclease domain fails to complement {Delta}polA. Cells lacking FEN ({Delta}fenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model is similar to models for Okazaki fragment processing in eukaryotes, where Pol d catalyzes strand displacement synthesis followed by 5' flap cleavage using FEN-1. Author Summary5' flap endo/exonuclease (FEN) activity provides an essential contribution to DNA replication and repair in all cellular life. In bacteria, DNA polymerase I is thought to be the central enzyme involved in Okazaki fragment processing, using its DNA polymerase and 5' nuclease activities to generate and then remove the 5 ssRNA segment of an Okazaki fragment. Many bacterial genomes encode a second, discrete FEN in addition to Pol I. We show that FEN is the primary 5' nuclease used by B. subtilis for primer removal. FEN activity exceeds that of Pol I on most substrates, including several that mimic Okazaki fragment intermediates. Additionally, we provide genetic evidence showing that FEN is involved in Okazaki fragment processing and that it is the DNA polymerase domain of Pol I rather than its 5' nuclease domain that is important in vivo. With our results, we propose a new model for Okazaki fragment processing in B. subtilis, which may be prevalent in a wider group of bacteria.

biochemistry↗