Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast

DNA polymerase epsilon (Pol ε) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3⁺ and dpb4⁺ that encode proteins homologous to the two smallest subunits of Pol ε. Although Dpb4 is not required for cell viability, Δdpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20⁺ (encoding DNA Pol ε), cut5⁺ (homologous to DPB11/TopBP1), sna41⁺ (homologous to CDC45) and cdc21⁺ (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S-transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol ε complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4.     

Structure and function of the fourth subunit (Dpb4p) of DNA polymerase epsilon in Saccharomyces cerevisiae

DNA polymerase (Pol) of Saccharomyces cerevisiae is purified as a complex of four polypeptides with molecular masses of >250, 80, 34 (and 31) and 29 kDa as determined by SDS–PAGE. The genes POL2, DPB2 and DPB3, encoding the catalytic Pol2p, the second (Dpb2p) and the third largest subunits (Dpb3p) of the complex, respectively, were previously cloned and characterised. This paper reports the partial amino acid sequence of the fourth subunit (Dpb4p) of Pol. This protein sequence matches parts of the predicted amino acid sequence from the YDR121w open reading frame on S.cerevisiae chromosome IV. Thus, YDR121w was renamed DPB4. A deletion mutant of DPB4 (Δdpb4) is not lethal, but chromosomal DNA replication is slightly disturbed in this mutant. A double mutant haploid strain carrying the Δdpb4 deletion and either pol2-11 or dpb11-1 is lethal at all temperatures tested. Furthermore, the restrictive temperature of double mutants carrying Δdpb4 and dpb2-1, rad53-1 or rad53-21 is lower than in the corresponding single mutants. These results strongly suggest that Dpb4p plays an important role in maintaining the complex structure of Pol in S.cerevisiae, even if it is not essential for cell growth. Structural homologues of DPB4 are present in other eukaryotic genomes, suggesting that the complex structure of S.cerevisiae Pol is conserved in eukaryotes.     

Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae

Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3' --> 5' proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 3' --> 5' exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.     

Sld2, Which Interacts with Dpb11 in Saccharomyces cerevisiae, Is Required for Chromosomal DNA Replication

The DPB11 gene, which genetically interacts with DNA polymerase II (), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.     

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.     

Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.     

Contributions of the individual hydrophobic clefts of the Escherichia coli β sliding clamp to clamp loading, DNA replication and clamp recycling

The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (β^C) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β⁺), and one β^C subunit (β⁺/β^C). The β⁺/β^C heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β⁺. Moreover, β⁺/β^C interacted normally with Pol III, and stimulated replication to the same extent as did β⁺. Finally, β⁺/β^C was severely impaired for unloading from DNA using either DnaX or the δ subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.     

Enzymatic switching for efficient and accurate translesion DNA replication

When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) δ or ε, a switch occurs to allow translesion synthesis by DNA polymerase η, followed by another switch that allows normal replication to resume. In the present study, we investigate these switches using Saccharomyces cerevisiae Pol δ, Pol ε and Pol η and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis–syn thymine–thymine (TT) dimer. We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol η and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.     

The Pol β-14 Dominant Negative Rat DNA Polymerase β Mutator Mutant Commits Errors during the Gap-Filling Step of Base Excision Repair in Saccharomyces cerevisiae

We demonstrated recently that dominant negative mutants of rat DNA polymerase β (Pol β) interfere with repair of alkylation damage in Saccharomyces cerevisiae. To identify the alkylation repair pathway that is disrupted by the Pol β dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol β mutants to genes known to be involved in repair of DNA alkylation damage in S. cerevisiae. We demonstrate that the rat Pol β mutants interfere with the base excision repair pathway in S. cerevisiae. In addition, expression of one of the Pol β dominant negative mutants, Pol β-14, increases the spontaneous mutation rate of S. cerevisiae whereas expression of another Pol β dominant negative mutant, Pol β-TR, does not. Expression of the Pol β-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate. These results suggest that gaps are required for mutagenesis to occur in the presence of Pol β-14 but that it is not merely the presence of a gap that results in mutagenesis. Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.     

Proteolysis of the Human DNA Polymerase Delta Smallest Subunit p12 by μ-Calpain in Calcium-Triggered Apoptotic HeLa Cells

Degradation of p12 subunit of human DNA polymerase delta (Pol δ) that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi), restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.     

Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant

The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (−1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the τ subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of τ, essential for interaction of τ with the α (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered α-τ interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of τ subunit, in securing a high fidelity of replication.     

In Vivo Protein Interactions within the Escherichia coli DNA Polymerase III Core

The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ ( subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 and dnaQ926 mutations do not affect the subunit-α subunit and subunit-θ subunit interactions. However, the dnaQ49 mutation greatly reduces the strength of interaction of the subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the protein, which leads to altered interactions within the Pol III core.     

Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase ζ

To probe Pol ζ functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol ζ in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol ζ is 30% as active as wild-type Pol ζ when replicating undamaged DNA. L979F Pol ζ shares with wild-type Pol ζ the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol ζ is error-prone compared to wild-type Pol ζ, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol ζ in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol ζ also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol ζ, and perhaps wild-type Pol ζ, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo.     

The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit

DNA polymerase δ (Pol δ) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination. In fission yeast, Pol δ is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1. Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex. Here it is shown that removal of the tandem zinc finger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the C-terminal zinc finger module ZnF2 is both necessary and sufficient for binding to the B-subunit in vivo and in vitro. Extensive mutagenesis of the ZnF2 module identifies important residues for B-subunit binding. In particular, disruption of the ZnF2 module by substitution of the putative metal-coordinating cysteines with alanine abolishes B-subunit binding and in vivo function. Finally, evidence is presented suggesting that the ZnF region is post-translationally modified in fission yeast cells.     

A four-subunit DNA polymerase ζ complex containing Pol δ accessory subunits is essential for PCNA-mediated mutagenesis

DNA polymerase ζ (Pol ζ) plays a key role in DNA translesion synthesis (TLS) and mutagenesis in eukaryotes. Previously, a two-subunit Rev3–Rev7 complex had been identified as the minimal assembly required for catalytic activity in vitro. Herein, we show that Saccharomyces cerevisiae Pol ζ binds to the Pol31 and Pol32 subunits of Pol δ, forming a four-subunit Pol ζ₄ complex (Rev3–Rev7–Pol31–Pol32). A [4Fe-4S] cluster in Rev3 is essential for the formation of Pol ζ₄ and damage-induced mutagenesis. Pol32 is indispensible for complex formation, providing an explanation for the long-standing observation that pol32Δ strains are defective for mutagenesis. The Pol31 and Pol32 subunits are also required for proliferating cell nuclear antigen (PCNA)-dependent TLS by Pol ζ as Pol ζ₂ lacks functional interactions with PCNA. Mutation of the C-terminal PCNA-interaction motif in Pol32 attenuates PCNA-dependent TLS in vitro and mutagenesis in vivo. Furthermore, a mutant form of PCNA, encoded by the mutagenesis-defective pol30-113 mutant, fails to stimulate Pol ζ₄ activity, providing an explanation for the observed mutagenesis phenotype. A stable Pol ζ₄ complex can be identified in all phases of the cell cycle suggesting that this complex is not regulated at the level of protein interactions between Rev3-Rev7 and Pol31-Pol32.     

The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA

Saccharomyces cerevisiae DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol δ and Pol ε in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol ε has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol δ has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol δ and Pol ε was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol δ and Pol ε on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol ε when compared to Pol δ. We conclude that Pol ε and Pol δ exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.     

Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to >95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.     

A Novel Function of CRL4Cdt2: REGULATION OF THE SUBUNIT STRUCTURE OF DNA POLYMERASE δ IN RESPONSE TO DNA DAMAGE AND DURING THE S PHASE*

DNA polymerase δ (Pol δ4) is a heterotetrameric enzyme, whose p12 subunit is degraded in response to DNA damage, leaving behind a trimer (Pol δ3) with altered enzymatic characteristics that participate in gap filling during DNA repair. We demonstrate that CRL4^Cdt2, a key regulator of cell cycle progression that targets replication licensing factors, also targets the p12 subunit of Pol δ4 in response to DNA damage and on entry into S phase. Evidence for the involvement of CRL4^Cdt2 included demonstration that p12 possesses a proliferating cell nuclear antigen-interacting protein-degron (PIP-degron) and that knockdown of the components of the CRL4^Cdt2 complex inhibited the degradation of p12 in response to DNA damage. Analysis of p12 levels in synchronized cell populations showed that p12 is partially degraded in S phase and that this is affected by knockdowns of CUL4A or CUL4B. Laser scanning cytometry of overexpressed wild type p12 and a mutant resistant to degradation showed that the reduction in p12 levels during S phase was prevented by mutation of p12. Thus, CRL4^Cdt2 also regulates the subunit composition of Pol δ during the cell cycle. These studies reveal a novel function of CRL4^Cdt2, i.e. the direct regulation of DNA polymerase δ, adding to its known functions in the regulation of the licensing of replication origins and expanding the scope of its overall control of DNA replication. The formation of Pol δ3 in S phase as a normal aspect of cell cycle progression leads to the novel implications that it is involved in DNA replication as well as DNA repair.     

Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR

The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4–M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4–M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function.