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1. The translesion polymerase Pol Y1 is a constitutive component of the B. subtilis replication machinery

   https://doi.org/10.1093/nar/gkae637  

   Marrin, McKayla E; Foster, Michael R; Santana, Chloe M; Choi, Yoonhee; Jassal, Avtar S; Rancic, Sarah J; Greenwald, Carolyn R; Drucker, Madeline N; Feldman, Denholm T; Thrall, Elizabeth S (July 2024, Nucleic Acids Research)

   Abstract Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacterium Escherichia coli has served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacterium Bacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to its E. coli homolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species. 

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2. Elevated Levels of the Escherichia coli nrdAB -Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the β Clamp

   https://doi.org/10.1128/JB.00304-21  

   Babu, Vignesh M.; Homiski, Caleb; Scotland, Michelle K.; Chodavarapu, Sundari; Kaguni, Jon M.; Sutton, Mark D. (November 2021, Journal of Bacteriology)

   Silhavy, Thomas J. (Ed.)

   ABSTRACT Expression of the Escherichia coli dnaN -encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (β E202K bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaN E202K strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB , that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the β clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaN E202K allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the β E202K and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB -encoded RNR relieved the growth defect caused by elevated levels of the β clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated β clamp levels to impede growth and suggest either that multiple effects stemming from the dnaN E202K mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the β clamp-Hda complex. The dnaN E202K allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaN E202K strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and β E202K clamp proteins support equivalent levels of Hda activity in vitro . Taken together, these results suggest that β E202K -Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed. 

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3. The Escherichia coli clamp loader rapidly remodels SSB on DNA to load clamps

   https://doi.org/10.1093/nar/gkac1169  

   Newcomb, Elijah S; Douma, Lauren G; Morris, Leslie A; Bloom, Linda B (December 2022, Nucleic Acids Research)

   Abstract Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme–DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein–protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein–protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading. 

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4. The Mutant β ^E202K Sliding Clamp Protein Impairs DNA Polymerase III Replication Activity

   https://doi.org/10.1128/JB.00303-21  

   Homiski, Caleb; Scotland, Michelle K.; Babu, Vignesh M.; Chodavarapu, Sundari; Maul, Robert W.; Kaguni, Jon M.; Sutton, Mark D. (November 2021, Journal of Bacteriology)

   Silhavy, Thomas J. (Ed.)

   ABSTRACT Expression of the Escherichia coli dnaN -encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro . Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the β E202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still sequester Pol III effectively. Of interest, β E202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaN E202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant β E202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that β E202K is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV. 

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5. ParB C-terminal lysine residues are essential for dimerization, in vitro DNA sliding and in vivo function

   https://doi.org/10.1101/2025.06.10.659001  

   Aleshintsev, Aleksey; Way, Linsey E; Guerra, Bianca; Serwaa, Lois Akosua; Molina, Miranda; Wang, Xindan; Kim, HyeongJun (June 2025, bioRxiv)

   ABSTRACT The broadly conserved ParB protein performs crucial functions in bacterial chromosome segregation and replication regulation. The cellular function of ParB requires it to dimerize, recognizeparSDNA sequences, clamp on DNA, then slide to adjacent sequences through nonspecific DNA binding. How ParB coordinates nonspecific DNA binding and sliding remains elusive. Here, we combine multiplein vitrobiophysical and computational tools andin vivoapproaches to address this question. We found that the five conserved lysine residues in the C-terminal domain of ParB play distinct roles in proper positioning and sliding on DNA, and their integrity is crucial for ParB’sin vivofunctions. Many proteins with diverse cellular activities need to move on DNA while loosely bound. Our findings reveal the detailed molecular mechanism by which multiple flexible basic residues enable DNA binding proteins to efficiently slide along DNA. 

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