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1. Deciphering the structural consequences of R83 and R152 methylation on DNA polymerase β using molecular modeling

   https://doi.org/10.1371/journal.pone.0318614  

   Srivastava, Amit; Idriss, Haitham; Das, Gobind; Abedrabbo, Sufian; Shamsir, Mohd Sahir; Homouz, Dirar (March 2025, PLOS ONE)

   Selvaraj, Chandrabose (Ed.)

   DNA polymerase β, a member of the X-family of DNA polymerases, undergoes complex regulations both in vitro and in vivo through various posttranslational modifications, including phosphorylation and methylation. The impact of these modifications varies depending on the specific amino acid undergoing alterations. In vitro, methylation of DNA polymerase β with the enzyme protein arginine methyltransferase 6 (PRMT6) at R83 and R152 enhances polymerase activity by improving DNA binding and processivity. Although these studies have shown that methylation improves DNA binding, the underlying mechanism of enhancement of polymerase activity in terms of structure and dynamics remains poorly understood. To address this gap, we modeled the methylated enzyme/DNA complex and conducted a microsecond-long simulation in the presence of Mg ions. Our results revealed significant structural changes induced by methylating both R83 and R152 sites in the enzyme. Specifically, these changes caused the DNA fragment to move closer to the C- and N-subdomains, forming additional hydrogen bonds. Furthermore, the cross-correlation map demonstrated that methylation enhanced long-range correlations within the domains/subdomains of DNA polymerase β, along with an increase in the linear mutual information value between the domains/subdomains and DNA fragments. The graph connectivity network also illustrated that methylation modulates the information pathway and identifies residues exhibiting long-distance coupling with the methylated sites. Our results provide an atomic-level understanding of the structural transition induced by methylation, shedding light on the mechanisms underlying the methylation-induced enhancement of activity in DNA polymerase β. 

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2. An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ

   https://doi.org/10.1126/sciadv.adl3214  

   Park, Joon; Herrmann, Geoffrey K; Roy, Arkanil; Shumate, Christie K; Cisneros, G Andrés; Yin, Y Whitney (May 2024, Science Advances)

   The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2′-deoxycytidine 5′-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3′-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase. 

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3. 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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4. 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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5. DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation

   https://doi.org/10.3390/genes14040774  

   Njeri, Catherine; Pepenella, Sharon; Battapadi, Tripthi; Bambara, Robert A; Balakrishnan, Lata (April 2023, Genes)

   DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication. 

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