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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. Elf1 promotes Rad26’s interaction with lesion-arrested Pol II for transcription-coupled repair

   https://doi.org/10.1073/pnas.2314245121  

   Sarsam, Reta D; Xu, Jun; Lahiri, Indrajit; Gong, Wenzhi; Li, Qingrong; Oh, Juntaek; Zhou, Zhen; Hou, Peini; Chong, Jenny; Hao, Nan; et al (January 2024, Proceedings of the National Academy of Sciences)

   Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a common mechanism to recognize a stalled Pol II, with additional interactions when Pol II is arrested at a lesion. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces further interactions between Rad26 and a lesion-arrested Pol II. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation. Together, our results provide important mechanistic insights into how two conserved transcription-coupled repair factors, Rad26/CSB and Elf1/ELOF1, work together at the initial lesion recognition steps of transcription-coupled repair. 

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3. RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21

   https://doi.org/10.3390/cancers14225544  

   Jacobs, Ruth Q.; Fuller, Kaila B.; Cooper, Stephanie L.; Carter, Zachariah I.; Laiho, Marikki; Lucius, Aaron L.; Schneider, David A. (November 2022, Cancers)

   Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols’ elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I. 

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4. Events associated with DNA replication disruption are not observed in hydrogen peroxide-treated Escherichia coli

   https://doi.org/10.1093/g3journal/jkab044  

   Hoff, Chettar A; Schmidt, Sierra S; Hackert, Brandy J; Worley, Travis K; Courcelle, Justin; Courcelle, Charmain T (April 2021, G3 Genes|Genomes|Genetics)

   Rhind, N (Ed.)

   Abstract UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions. 

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5. The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

   https://doi.org/10.3390/cells10051083  

   Sikand, Adhirath; Jaszczur, Malgorzata; Bloom, Linda B.; Woodgate, Roger; Cox, Michael M.; Goodman, Myron F. (May 2021, Cells)

   null (Ed.)

   In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin. 

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