Cloning and Characterization of the Human Mitochondrial DNA Polymerase, DNA Polymerase γ

The nuclear-encoded DNA polymerase γ (DNA POLγ) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POLγ and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POLγ gene fromDrosophila melanogasterand a partial cDNA for DNA POLγ fromGallus gallushave been cloned. The predicted human DNA POLγ polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those ofSchizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster,and the C-terminal half ofG. gallus,respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POLγ like activity from mitochondrial extracts. The human DNA POLγ is unique in that the first exon of the gene contains a CAG₁₀trinucleotide repeat.     

Substrate-induced DNA Polymerase β Activation

DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase β, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.     

An Iron–Sulfur Cluster in the Polymerase Domain of Yeast DNA Polymerase ε

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits—Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1–1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2^ins) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.     

The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pulldown assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, whereas the whole catalytically active heterodimer of polα (p180ΔN-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180ΔN-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180ΔN-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70, and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homopyrimidine runs create a strong barrier for DNA synthesis by polα.     

RNA Polymerase Activity Associated with Bacteriophage φ6

The Pseudomonas phaseolicola bacteriophage phi6 incorporated labeled UTP into an acid-insoluble precipitate. Incorporation was dependent on the presence of manganese acetate, ATP, GTP, CTP, and a short heat treatment of the phage; the reaction was stimulated by NH(4)Cl. The substitution of (14)C-ATP, -CTP or -GTP for UTP, together with the appropriate unlabeled ribonucleoside triphosphates, disclosed that CMP was incorporated to the greatest extent followed by GMP, UMP, and AMP. Radioactive RNAs formed by the reaction were resistant to RNases A and T(1) in high salt but susceptible to these nucleases in low salt. The labeled RNA co-sedimented and co-electrophoresed with phi6 double-stranded (ds) RNA. However, the distribution of the radioactivity into the three ds-RNA components varied depending on the (14)C-ribonucleoside triphosphate used in the reaction. The incorporation of UMP was primarily into the two smaller ds-RNA segments, GMP primarily into the large ds-RNA segment, and CMP and AMP were about equally distributed into all three ds-RNA segments.     

Interaction between DNA Polymerase β and BRCA1

The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.     

DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.     

聚合酶链反应（Polymerase Chain Reaction，PCR）

PCR是80年代中期发展起来的体外核酸扩增技术。它具有特异、敏感、产率高、快速、简便、重复性好、易自动化等突出的优点;能在一个试管内将所要研究的目的基因或某一DNA片段于数小时内扩增至十万乃至百万倍,使肉眼能直接观察和判断;可从一根毛发、一滴血、甚至一个细胞中扩增出足量的DNA供分析研究和检测鉴定。过去几天几星期才能做到的事情,用PCR几小时便可完成。PCR技术是生物医学领域中的一项革命性创举和里程碑。     

Alternative Mechanisms for Coordinating Polymerase α and MCM Helicase

Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase α in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polα. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polα without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polα at the elongating replication fork—first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.The key players of the replication machinery are the DNA polymerases that synthesize the leading and lagging daughter strands and the replicative helicase that unwinds the parental strands ahead of the polymerases. Coordination between the helicase and the polymerases is critical during replication. Uncoupling of these two molecular machines, especially during lagging strand synthesis, may result in an unrestrained helicase and the exposure of extensive single-stranded DNA (ssDNA), as observed in checkpoint mutants treated with hydroxyurea (HU) (37). Although there is no direct evidence, the implication is that the replicative helicase would be moving at a faster pace than would the DNA polymerase if synchrony were destroyed. In Escherichia coli, the replicative helicase (DnaB) and the primase (DnaG) are coupled by direct contact to form a tight complex (3). In T7, processivity of the gp5 polymerase in lagging strand synthesis requires coupling to the gp4 helicase (16). Recent studies of the budding yeast Saccharomyces cerevisiae suggest that Mrc1 may couple DNA polymerase ɛ and the MCM helicase on the leading strand as well as activate the checkpoint response under replication stress (1, 22, 28). A candidate for coupling DNA polymerase α primase and the MCM helicase on the lagging strand is Mcm10, because Mcm10 interacts with subunits of the Mcm2-7 helicase (26, 29) as well as Polα (14, 33) and the stability of Polα requires Mcm10 in both budding yeast and human cells (8, 33). Mcm10 is an essential protein known to be involved in various aspects of the replication process. It is required during both initiation and elongation steps of DNA replication and interacts with a wide range of replication factors, such as ORC (17, 23, 29), MCM helicase, DNA polymerases ɛ and δ (23), Cdc45 (34), and Polα (33). Therefore, Mcm10 is important for the overall stability of the elongation complex, but its essential function remains unknown.Accumulating evidence suggests that the major function of many checkpoint proteins is the stabilization of the replication machinery at the fork (9, 22, 39), in addition to regulation of the temporal and spatial firing of origins and prevention of premature mitosis (31, 35, 39). The main signal that leads to checkpoint activation is believed to be the exposure of RPA-coated ssDNA (42). In Xenopus, ssDNA exposure has been shown to be mediated by a functional uncoupling between the polymerase and the helicase (7), and it has been shown that the level of checkpoint activation depended on the extent of ssDNA accumulation. This observation suggests that uncoupling of the polymerase and the helicase activity would result in ssDNA accumulation that in turn would activate the checkpoint pathway to stabilize the fork.In our study, we carried out a random and a gene-targeted mutagenesis screen to identify mutations that suppress the conditional lethality of mcm10 caused by the lability of Mcm10 in budding yeast (27). We found suppressor mutations in MCM2, which encodes one of the six distinct subunits of the MCM helicase. These mcm2 mutations correct the fork defects of mcm10, particularly that which leads to Polα instability. The altered helicase activity and activation of the checkpoint pathway of the mcm2 mutants appeared to be required for viability of mcm10 mcm2. We showed that uncoupling the MCM helicase and DNA polymerase α by destabilizing Mcm10 leads to accumulation of ssDNA, which is suppressed by reducing the MCM helicase activity. Our findings suggest that the physical coupling of Polα and the helicase by Mcm10 may be replaced by an alternative stabilization mechanism that involves slowing down the helicase and activating the checkpoint proteins.     

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase λ (Pol λ) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol λ. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol λ efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol λ could modulate the cytotoxic effect of AraC.     

Crystal Structure of Yeast DNA Polymerase ε Catalytic Domain

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2_G:C). Pol2_G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2_T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca²⁺ ions at both positions A and B in the polymerase active site and also observe a Ca²⁺ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2_G:C structure are maintained in the Pol2_T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2_T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.     

Comparative Analysis of Eubacterial DNA Polymerase Ⅲ Alpha Subunits

DNA polymerase Ⅲ is one of the five eubacterial DNA polymerases that is responsible for the replication of DNA duplex. Among the ten subunits of the DNA polymerase Ⅲ core enzyme, the alpha subunit catalyzes the reaction for polymerizing both DNA strands. In this study, we extracted genomic sequences of the alpha subunit from 159 sequenced eubacterial genomes, and carried out sequence- based phylogenetic and structural analyses. We found that all eubacterial genomes have one or more alpha subunits, which form either homodimers or heterodimers. Phylogenetic and domain structural analyses as well as copy number variations of the alpha subunit in each bacterium indicate the classification of alpha subunit into four basic groups： polC, dnaE1, dnaE2, and dnaE3. This classification is of essence in genome composition analysis. We also consolidated the naming convention to avoid further confusion in gene annotations.     

Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase β

Background

Altered expression of DNA polymerase β (Pol β) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol β over-expression has not yet been evaluated in a mouse model.

Methodology/Principal Findings

We have recently developed a novel transgenic mouse model that over-expresses Pol β. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner''s gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol β over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol β expression. We observed elevated expression of Pol β in stomach adenomas and thyroid follicular carcinomas, but reduced Pol β expression in esophageal adenocarcinomas and squamous carcinomas.

Conclusions/Significance

These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.     

The Human Lagging Strand DNA Polymerase δ Holoenzyme Is Distributive

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5′-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ^AA. The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.     

Translesion Synthesis of Abasic Sites by Yeast DNA Polymerase ?

Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase ϵ bypasses a model abasic site with comparable efficiency to Pol η and Dpo4, two translesion polymerases. DNA polymerase ϵ also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase ϵ with 3′–5′ proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.     

Polymerase η suppresses telomere defects induced by DNA damaging agents

Telomeres at chromosome ends are normally masked from proteins that signal and repair DNA double strand breaks (DSBs). Bulky DNA lesions can cause DSBs if they block DNA replication, unless they are bypassed by translesion (TLS) DNA polymerases. Here, we investigated roles for TLS polymerase η, (polη) in preserving telomeres following acute physical UVC exposure and chronic chemical Cr(VI) exposure, which both induce blocking lesions. We report that polη protects against cytotoxicity and replication stress caused by Cr(VI), similar to results with ultraviolet C light (UVC). Both exposures induce ataxia telangiectasia and Rad3-related (ATR) kinase and polη accumulation into nuclear foci and localization to individual telomeres, consistent with replication fork stalling at DNA lesions. Polη-deficient cells exhibited greater numbers of telomeres that co-localized with DSB response proteins after exposures. Furthermore, the genotoxic exposures induced telomere aberrations associated with failures in telomere replication that were suppressed by polη. We propose that polη''s ability to bypass bulky DNA lesions at telomeres is critical for proper telomere replication following genotoxic exposures.     

Coupling σ Factor Conformation to RNA Polymerase Reorganisation for DNA Melting

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (σ⁵⁴-RNAP, Eσ⁵⁴) and a slowly hydrolysed ATP analogue (ATPγS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of σ⁵⁴-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Eσ⁵⁴ occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity σ⁵⁴ factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic β/β′ subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Eσ⁵⁴ promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the β′ jaw domain, thereby allowing acquisition of the open complex status.