Whole-cell bioconversion for producing thymoquinone by engineered Saccharomyces cerevisiae.

Thymoquinone, extracted from the black seeds of Nigella sativa, is a natural substance with highly beneficial effects against various human diseases. In this study, we aimed to construct a Saccharomyces cerevisiae strain that, produce thymoquinone from thymol, a relatively inexpensive substrate. To achieve this, cytochrome P450 from Origanum vulgare was expressed in S. cerevisiae for the bioconversion of thymol to thymoquinone, with the co-expression of cytochrome P450 reductase (CPR) from Arabidopsis thaliana, ATR1. Additionally, flexible linkers were used to connect these two enzymes. Furthermore, modifications were performed to expand the endoplasmic reticulum (ER) space, leading to increased thymoquinone production. After integrating the genes into the chromosome and optimizing the media components, a significant improvement in the thymol-to-thymoquinone conversion rate and yield were achieved. This study represents a possibility of the production of thymoquinone, a bioactive ingredient of a plant, using an engineered microbial cell.

Molecular choreography: Unveiling the dynamic landscape of type IIA DNA topoisomerases before T-segment passage through all-atom simulations.

Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations, and performed all-atom simulations. The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.

Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Transcription elongation mechanisms of RNA polymerases I, II, and III and their therapeutic implications.

Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.

Structure, cooperativity and inhibition of the inosine 5'-monophosphate-specific phosphatase from Saccharomyces cerevisiae.

The nucleoside inosine is a main intermediate of purine nucleotide catabolism in Saccharomyces cerevisiae and is produced via the dephosphorylation of inosine monophosphate (IMP) by IMP-specific 5'-nucleotidase 1 (ISN1), which is present in many eukaryotic organisms. Upon transition of yeast from oxidative to fermentative growth, ISN1 is important for intermediate inosine accumulation as purine storage, but details of ISN1 regulation are unknown. We characterized structural and kinetic behavior of ISN1 from S. cerevisiae (ScISN1) and showed that tetrameric ScISN1 is negatively regulated by inosine and adenosine triphosphate (ATP). Regulation involves an inosine-binding allosteric site along with IMP-induced local and global conformational changes in the monomer and a tetrameric re-arrangement, respectively. A proposed interaction network propagates local conformational changes in the active site to the intersubunit interface, modulating the allosteric features of ScISN1. Via ATP and inosine, ScISN1 activity is likely fine-tuned to regulate IMP and inosine homeostasis. These regulatory and catalytic features of ScISN1 contrast with those of the structurally homologous ISN1 from Plasmodium falciparum, indicating that ISN1 enzymes may serve different biological purposes in different organisms.

Secretory Production of the Hericium erinaceus Laccase from Saccharomyces cerevisiae.

Mushroom laccases play a crucial role in lignin depolymerization, one of the most critical challenges in lignin utilization. Importantly, laccases can utilize a wide range of substrates, such as toxicants and antibiotics. This study isolated a novel laccase, named HeLac4c, from endophytic white-rot fungi Hericium erinaceus mushrooms. The cDNAs for this enzyme were 1569 bp in length and encoded a protein of 523 amino acids, including a 20 amino-acid signal peptide. Active extracellular production of glycosylated laccases from Saccharomyces cerevisiae was successfully achieved by selecting an optimal translational fusion partner. We observed that 5 and 10 mM Ca2+, Zn2+, and K+ increased laccase activity, whereas 5 mM Fe2+ and Al3+ inhibited laccase activity. The laccase activity was inhibited by the addition of low concentrations of sodium azide and L-cysteine. The optimal pH for the 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was 4.4. Guaiacylglycerol-ß-guaiacyl ether, a lignin model compound, was polymerized by the HeLac4c enzyme. These results indicated that HeLac4c is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products for environmental biotechnological applications.

Catalytic mechanism of trans-2-enoyl-CoA reductases in the fatty acid elongation cycle and its cooperative action with fatty acid elongases.

The fatty acid (FA) elongation cycle produces very-long-chain FAs with ≥C21, which have unique physiological functions. Trans-2-enoyl-CoA reductases (yeast, Tsc13; mammals, TECR) catalyze the reduction reactions in the fourth step of the FA elongation cycle and in the sphingosine degradation pathway. However, their catalytic residues and coordinated action in the FA elongation cycle complex are unknown. To reveal these, we generated and analyzed Ala-substituted mutants of 15 residues of Tsc13. An in vitro FA elongation assay showed that nine of these mutants were less active than WT protein, with E91A and Y256A being the least active. Growth complementation analysis, measurement of ceramide levels, and deuterium-sphingosine labeling revealed that the function of the E91A mutant was substantially impaired in vivo. In addition, we found that the activity of FA elongases, which catalyze the first step of the FA elongation cycle, were reduced in the absence of Tsc13. Similar results were observed in Tsc13 E91A-expressing cells, which is attributable to reduced interaction between the Tsc13 E91A mutant and the FA elongases Elo2/Elo3. Finally, we found that E94A and Y248A mutants of human TECR, which correspond to E91A and Y256A mutants of Tsc13, showed reduced and almost no activity, respectively. Based on these results and the predicted three-dimensional structure of Tsc13, we speculate that Tyr256/Tyr248 of Tsc13/TECR is the catalytic residue that supplies a proton to trans-2-enoyl-CoAs. Our findings provide a clue concerning the catalytic mechanism of Tsc13/TECR and the coordinated action in the FA elongation cycle complex.

Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro.

Thiolutin is a natural product transcription inhibitor with an unresolved mode of action. Thiolutin and the related dithiolopyrrolone holomycin chelate Zn2+ and previous studies have concluded that RNA Polymerase II (Pol II) inhibition in vivo is indirect. Here, we present chemicogenetic and biochemical approaches to investigate thiolutin's mode of action in Saccharomyces cerevisiae. We identify mutants that alter sensitivity to thiolutin. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo and that thiolutin both induces oxidative stress and interacts functionally with multiple metals including Mn2+ and Cu2+, and not just Zn2+. Finally, we show direct inhibition of RNA polymerase II (Pol II) transcription initiation by thiolutin in vitro in support of classical studies that thiolutin can directly inhibit transcription in vitro. Inhibition requires both Mn2+ and appropriate reduction of thiolutin as excess DTT abrogates its effects. Pause prone, defective elongation can be observed in vitro if inhibition is bypassed. Thiolutin effects on Pol II occupancy in vivo are widespread but major effects are consistent with prior observations for Tor pathway inhibition and stress induction, suggesting that thiolutin use in vivo should be restricted to studies on its modes of action and not as an experimental tool.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Human V-ATPase a-subunit isoforms bind specifically to distinct phosphoinositide phospholipids.

Vacuolar H+-ATPases (V-ATPases) are highly conserved multisubunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue- and organelle-specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle-specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms, and we hypothesize that the aNT domains of these isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. We determined that bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP-binding sites, we identified potential binding sites in the HuaNT domains by sequence comparisons and existing subunit structures and models. We found that mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles.

A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides.

The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2'-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.

Structures illustrate step-by-step mitochondrial transcription initiation.

Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.

Turnover and bypass of p21-activated kinase during Cdc42-dependent MAPK signaling in yeast.

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular behaviors, including the response to stress and cell differentiation, and are highly conserved across eukaryotes. MAPK pathways can be activated by the interaction between the small GTPase Cdc42p and the p21-activated kinase (Ste20p in yeast). By studying MAPK pathway regulation in yeast, we recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is regulated in a similar manner and is turned over by the 26S proteasome. This turnover did not occur when Ste20p was bound to Cdc42p, which presumably stabilized the protein to sustain MAPK pathway signaling. Although Ste20p is a major component of the fMAPK pathway, genetic approaches here identified a Ste20p-independent branch of signaling. Ste20p-independent signaling partially required the fMAPK pathway scaffold and Cdc42p-interacting protein, Bem4p, while Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p. Interestingly, Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p, Rga1p, which unexpectedly dampened basal but not active fMAPK pathway activity. These new regulatory features of the Rho GTPase and p21-activated kinase module may extend to related pathways in other systems.

Elongation rate of RNA polymerase II affects pausing patterns across 3' UTRs.

Yeast mRNAs are polyadenylated at multiple sites in their 3' untranslated regions (3' UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites, and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the effect of Pol II elongation rate on pausing patterns and the relationship between Pol II pause sites and poly(A) sites within 3' UTRs. Mutations that affect Pol II elongation rate alter sequence preferences at pause sites within 3' UTRs, and pausing preferences differ between 3' UTRs and coding regions. In addition, sequences immediately flanking the pause sites show preferences that are largely independent of Pol II speed. In wild-type cells, poly(A) sites are preferentially located < 50 nucleotides upstream from Pol II pause sites, but this spatial relationship is diminished in cells harboring Pol II speed mutants. Based on a random forest classifier, Pol II pause sites are modestly predicted by the distance to poly(A) sites but are better predicted by the chromatin landscape in Pol II speed derivatives. Transcriptional regulatory proteins can influence the relationship between Pol II pausing and polyadenylation but in a manner distinct from Pol II elongation rate derivatives. These results indicate a complex relationship between Pol II pausing and polyadenylation.

A conserved arginine within the αC-helix of Erk1/2 is a latch of autoactivation and of oncogenic capabilities.

Eukaryotic protein kinases (EPKs) adopt an active conformation following phosphorylation of a particular activation loop residue. Most EPKs spontaneously autophosphorylate this residue. While structure-function relationships of the active conformation are essentially understood, those of the "prone-to-autophosphorylate" conformation are unclear. Here, we propose that a site within the αC-helix of EPKs, occupied by Arg in the mitogen-activated protein kinase (MAPK) Erk1/2 (Arg84/65), impacts spontaneous autophosphorylation. MAPKs lack spontaneous autoactivation, but we found that converting Arg84/65 of Erk1/2 to various residues enables spontaneous autophosphorylation. Furthermore, Erk1 molecules mutated in Arg84 are oncogenic. Arg84/65 thus obstructs the adoption of the "prone-to-autophosphorylate" conformation. All MAPKs harbor an Arg that is equivalent to Arg84/65 of Erks, whereas Arg is rarely found at the equivalent position in other EPKs. We observed that Arg84/65 of Erk1/2 interacts with the DFG motif, suggesting that autophosphorylation may be inhibited by the Arg84/65-DFG interactions. Erk1/2s mutated in Arg84/65 autophosphorylate not only the TEY motif, known as critical for catalysis, but also on Thr207/188. Our MS/MS analysis revealed that a large proportion of the Erk2R65H population is phosphorylated on Thr188 or on Tyr185 + Thr188, and a small fraction is phosphorylated on the TEY motif. No molecules phosphorylated on Thr183 + Thr188 were detected. Thus, phosphorylation of Thr183 and Thr188 is mutually exclusive suggesting that not only TEY-phosphorylated molecules are active but perhaps also those phosphorylated on Tyr185 + Thr188. The effect of mutating Arg84/65 may mimic a physiological scenario in which allosteric effectors cause Erk1/2 activation by autophosphorylation.

An NMR Study of a 300-kDa AAA+ Unfoldase.

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

DNA polymerase ε leading strand signature mutations result from defects in its proofreading activity.

The evidence that purified pol2-M644G DNA polymerase (Pol)ε exhibits a highly elevated bias for forming T:dTTP mispairs over A:dATP mispairs and that yeast cells harboring this Polε mutation accumulate A > T signature mutations in the leading strand have been used to assign a role for Polε in replicating the leading strand. Here, we determine whether A > T signature mutations result from defects in Polε proofreading activity by analyzing their rate in Polε proofreading defective pol2-4 and pol2-M644G cells. Since purified pol2-4 Polε exhibits no bias for T:dTTP mispair formation, A > T mutations are expected to occur at a much lower rate in pol2-4 than in pol2-M644G cells if Polε replicated the leading strand. Instead, we find that the rate of A > T signature mutations are as highly elevated in pol2-4 cells as in pol2-M644G cells; furthermore, the highly elevated rate of A > T signature mutations is severely curtailed in the absence of PCNA ubiquitination or Polζ in both the pol2-M644G and pol2-4 strains. Altogether, our evidence supports the conclusion that the leading strand A > T signature mutations derive from defects in Polε proofreading activity and not from the role of Polε as a leading strand replicase, and it conforms with the genetic evidence for a major role of Polδ in replication of both the DNA strands.

Mismatch repair operates at the replication fork in direct competition with mismatch extension by DNA polymerase δ.

DNA mismatch repair (MMR) in eukaryotes is believed to occur post-replicatively, wherein nicks or gaps in the nascent DNA strand are suggested to serve as strand discrimination signals. However, how such signals are generated in the nascent leading strand has remained unclear. Here we examine the alternative possibility that MMR occurs in conjunction with the replication fork. To this end, we utilize mutations in the PCNA interacting peptide (PIP) domain of the Pol3 or Pol32 subunit of DNA polymerase δ (Polδ) and show that these pip mutations suppress the greatly elevated mutagenesis in yeast strains harboring the pol3-01 mutation defective in Polδ proofreading activity. And strikingly, they suppress the synthetic lethality of pol3-01 pol2-4 double mutant strains, which arises from the vastly enhanced mutability due to defects in the proofreading functions of both Polδ and Polε. Our finding that suppression of elevated mutagenesis in pol3-01 by the Polδ pip mutations requires intact MMR supports the conclusion that MMR operates at the replication fork in direct competition with other mismatch removal processes and with extension of synthesis from the mispair by Polδ. Furthermore, the evidence that Polδ pip mutations eliminate almost all the mutability of pol2-4 msh2Δ or pol3-01 pol2-4 adds strong support for a major role of Polδ in replication of both the leading and lagging DNA strands.