Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.     

The influence of the DNA polymerase gamma accessory subunit on base excision repair by the catalytic subunit

Mammalian DNA polymerase gamma, the sole polymerase responsible for replication and repair of mitochondrial DNA, contains a large catalytic subunit and a smaller accessory subunit, pol gammaB. In addition to the polymerase domain, the large subunit contains a 3'-5' editing exonuclease domain as well as a dRP lyase activity that can remove a 5'-deoxyribosephosphate (dRP) group during base excision repair. We show that the accessory subunit enhances the ability of the catalytic subunit to function in base excision repair mainly by stimulating two subreactions in the repair process. Pol gammaB appeared to specifically enhance the rate at which pol gamma was able to locate damage in high molecular weight DNA. One pol gammaB point mutant known to have impaired ability to bind duplex DNA stimulated repair poorly, suggesting that duplex DNA binding through pol gammaB may help the catalytic subunit locate sites of DNA damage. In addition, the small subunit significantly stimulated the dRP lyase activity of pol gammaA, although it did not increase the rate at which the dRP group dissociated from the enzyme. The ability of DNA pol gamma to process a high load of damaged DNA may be compromised by the slow release of the dRP group.     

Residues of human cytomegalovirus DNA polymerase catalytic subunit UL54 that are necessary and sufficient for interaction with the accessory protein UL44

The human cytomegalovirus DNA polymerase contains a catalytic subunit, UL54, and an accessory protein, UL44. Recent studies suggested that UL54 might interact via its extreme C terminus with UL44 (A. Loregian, R. Rigatti, M. Murphy, E. Schievano, G. Palu', and H. S. Marsden, J. Virol. 77:8336-8344, 2003). To address this hypothesis, we quantitatively measured the binding of peptides corresponding to the extreme C terminus of UL54 to UL44 by using isothermal titration calorimetry. A peptide corresponding to the last 22 residues of UL54 was sufficient to bind specifically to UL44 in a 1:1 complex with a dissociation constant of ca. 0.7 microM. To define individual residues in this segment that are crucial for interacting with UL44, we engineered a series of mutations in the C-terminal region of UL54. The UL54 mutants were tested for their ability to interact with UL44 by glutathione S-transferase pulldown assays, for basal DNA polymerase activity, and for long-chain DNA synthesis in the presence of UL44. We observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction. Thus, like the herpes simplex virus UL30-UL42 interaction, a few specific side chains in the C terminus of UL54 are crucial for UL54-UL44 interaction. However, the UL54 residues important for interaction with UL44 are hydrophobic and not basic. This information might aid in the rational design of new drugs for the treatment of human cytomegalovirus infection.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase,POLG1)

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.     

The accessory subunit B of DNA polymerase gamma is required for mitochondrial replisome function

The mitochondrial replication machinery in human cells includes the DNA polymerase γ holoenzyme and the TWINKLE helicase. Together, these two factors form a processive replication machinery, a replisome, which can use duplex DNA as template to synthesize long stretches of single-stranded DNA. We here address the importance of the smaller, accessory B subunit of DNA polymerase γ and demonstrate that this subunit is absolutely required for replisome function. The duplex DNA binding activity of the B subunit is needed for coordination of POLγ holoenzyme and TWINKLE helicase activities at the mtDNA replication fork. In the absence of proof for direct physical interactions between the components of the mitochondrial replisome, these functional interactions may explain the strict interdependence of TWINKLE and DNA polymerase γ for mitochondrial DNA synthesis. Furthermore, mutations in TWINKLE as well as in the catalytic A and accessory B subunits of the POLγ holoenzyme, may cause autosomal dominant progressive external ophthalmoplegia, a disorder associated with deletions in mitochondrial DNA. The crucial importance of the B subunit for replisome function may help to explain why mutations in these three proteins cause an identical syndrome.     

Specific residues in the connector loop of the human cytomegalovirus DNA polymerase accessory protein UL44 are crucial for interaction with the UL54 catalytic subunit

The human cytomegalovirus DNA polymerase includes an accessory protein, UL44, which has been proposed to act as a processivity factor for the catalytic subunit, UL54. How UL44 interacts with UL54 has not yet been elucidated. The crystal structure of UL44 revealed the presence of a connector loop analogous to that of the processivity subunit of herpes simplex virus DNA polymerase, UL42, which is crucial for interaction with its cognate catalytic subunit, UL30. To investigate the role of the UL44 connector loop, we replaced each of its amino acids (amino acids 129 to 140) with alanine. We then tested the effect of each substitution on the UL44-UL54 interaction by glutathione S-transferase pulldown and isothermal titration calorimetry assays, on the stimulation of UL54-mediated long-chain DNA synthesis by UL44, and on the binding of UL44 to DNA-cellulose columns. Substitutions that affected residues 133 to 136 of the connector loop measurably impaired the UL44-UL54 interaction without altering the ability of UL44 to bind DNA. One substitution, I135A, completely disrupted the binding of UL44 to UL54 and inhibited the ability of UL44 to stimulate long-chain DNA synthesis by UL54. Thus, similar to the herpes simplex virus UL30-UL42 interaction, a residue of the connector loop of the accessory subunit is crucial for UL54-UL44 interaction. However, while alteration of a polar residue of the UL42 connector loop only partially reduced binding to UL30, substitution of a hydrophobic residue of UL44 completely disrupted the UL54-UL44 interaction. This information may aid the discovery of small-molecule inhibitors of the UL44-UL54 interaction.     

In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.     

A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase

Mitochondrial DNA polymerase (pol gamma) is the sole DNA polymerase responsible for replication and repair of animal mitochondrial DNA. Here, we address the molecular mechanism by which the human holoenzyme achieves high processivity in nucleotide polymerization. We have determined the crystal structure of human pol gamma-beta, the accessory subunit that binds with high affinity to the catalytic core, pol gamma-alpha, to stimulate its activity and enhance holoenzyme processivity. We find that human pol gamma-beta shares a high level of structural similarity to class IIa aminoacyl tRNA synthetases, and forms a dimer in the crystal. A human pol gamma/DNA complex model was developed using the structures of the pol gamma-beta dimer and the bacteriophage T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol gamma-beta and the human catalytic core that allow it to encircle the newly synthesized double-stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Biochemical properties of a novel set of human pol gamma-beta mutants are explained by and test the model, and elucidate the role of the accessory subunit as a novel type of processivity factor in stimulating pol gamma activity and in enhancing processivity.     

The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.     

The fidelity of human DNA polymerase gamma with and without exonucleolytic proofreading and the p55 accessory subunit

Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we investigated the fidelity of DNA synthesis by human pol gamma. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol gamma has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol gamma is also relatively accurate for single-base additions and deletions in non-iterated and short repetitive sequences. However, when copying homopolymeric sequences longer than four nucleotides, pol gamma has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol gamma both to make and to proofread dislocation intermediates is the first such evidence for a family A polymerase. Including the p55 accessory subunit, which confers processivity to the pol gamma catalytic subunit, decreases frameshift and base substitution fidelity. Kinetic analyses indicate that p55 promotes extension of mismatched termini to lower the fidelity. These data suggest that homopolymeric runs in mitochondrial DNA may be particularly prone to frameshift mutation in vivo due to replication errors by pol gamma.     

The tamas gene, identified as a mutation that disrupts larval behavior in Drosophila melanogaster, codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-gamma125)

From a screen of pupal lethal lines of Drosophila melanogaster we identified a mutant strain that displayed a reproducible reduction in the larval response to light. Moreover, this mutant strain showed defects in the development of the adult visual system and failure to undergo behavioral changes characteristic of the wandering stage. The foraging third instar larvae remained in the food substrate for a prolonged period and died at or just before pupariation. Using a new assay for individual larval photobehavior we determined that the lack of response to light in these mutants was due to a primary deficit in locomotion. The mutation responsible for these phenotypes was mapped to the lethal complementation group l(2)34Dc, which we renamed tamas (translated from Sanskrit as "dark inertia"). Sequencing of mutant alleles demonstrated that tamas codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-gamma125).     

The 44P subunit of the T4 DNA polymerase accessory protein complex catalyzes ATP hydrolysis

The genes encoding all three T4 DNA polymerase accessory proteins have been cloned into overexpression plasmids. Induction of cells harboring these plasmids results in the synthesis of each accessory protein at levels that approach 10% of the total cellular protein. The solubility of the accessory proteins after induction at 42 degrees C ranges from about 60% to greater than 95%. A plasmid that allows overexpression of the 44P/62P complex has been manipulated further to overexpress selectively the 44P subunit without 62P, permitting us to assess how each subunit contributes to the properties of the 44P/62P complex. A comparison of 44P and 44P/62P by conventional hydrodynamic techniques shows that 44P forms a subcomplex nearly as large as the 44P/62P complex. In addition, 44P catalyzes DNA-dependent ATP hydrolysis with a specific activity similar to that of the 44P/62P ATPase. However, unlike the 44P/62P complex, the ATPase activity of 44P alone is only slightly stimulated by 45P. This suggests that one role of the 62P subunit is to facilitate a productive interaction of 44P and 45P.     

Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase δ subunit Cdc27

Background  

DNA polymerases α and δ play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase α (Pol α)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase δ (Pol δ) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase ε (Pol ε).     

Overexpression of the catalytic subunit of DNA polymerase gamma results in depletion of mitochondrial DNA in Drosophila melanogaster

The mechanisms involved in the regulation of mitochondrial DNA (mtDNA) replication, a process that is crucial for mitochondrial biogenesis, are not well understood. In this study, we evaluate the role of DNA polymerase gamma (pol gamma), the key enzyme in mtDNA replication, in both Drosophila cell culture and in developing flies. We report that overexpression of the pol gamma catalytic subunit (pol gamma-alpha) in cultured Schneider cells does not alter either the amount of mtDNA or the growth rate of the culture. The polypeptide is properly targeted to mitochondria, yet the large excess of pol gamma-alpha does not interfere with mtDNA replication under these conditions where the endogenous polypeptide is apparently present in amounts that exceed of the demand for its function in the cell. In striking contrast, overexpression of pol gamma-alpha at the same level in transgenic flies interferes with the mtDNA replication process, presumably by altering the mechanism of DNA synthesis, suggesting differential requirements for, and/or regulation of, mtDNA replication in Drosophila cell culture versus the developing organism. Overexpression of pol gamma-alpha in transgenic flies produces a significant depletion of mtDNA that causes a broad variety of phenotypic effects. These alterations range from pupal lethality to moderate morphological abnormalities in adults. depending on the level and temporal pattern of overexpression. Our results demonstrate that although cells may tolerate a variable amount of the pol gamma catalytic subunit under some conditions, its level may be critical in the context of the whole organism.     

Purification and identification of subunit structure of the human mitochondrial DNA polymerase.

The mitochondrial DNA polymerase of HeLa cells was purified 18,000-fold to near homogeneity. The purified polymerase cofractionated with two polypeptides that had molecular mass of 140 and 54 kDa. The 140-kDa subunit was specifically radiolabeled in a photoaffinity cross-linking assay and is most likely the catalytic subunit of the mitochondrial DNA polymerase. The purified enzyme exhibited properties that have been attributed to DNA polymerase gamma and shows a preference for replicating primed poly(pyrimidine) DNA templates in the presence of 0.5 mM MgCl2. As in the case of mitochondrial DNA polymerases from other animal cells, human DNA polymerase gamma cofractionated with a 3'----5' exonuclease activity. However, it has not been possible to determine if the two enzymatic activities reside in the same polypeptide. The exonuclease activity preferentially removes mismatched nucleotides from the 3' end of a duplex DNA and is not active toward DNA with matched 3' ends. These properties are consistent with the notion that the exonuclease activity plays a proofreading function in the replication of the organelle genome.     

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.