The p12 Subunit of Human Polymerase δ Modulates the Rate and Fidelity of DNA Synthesis (original) (raw)
Related papers
Journal of Biological Chemistry, 2001
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 ␥ (pol ␥), we investigated the fidelity of DNA synthesis by human pol ␥. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol ␥ has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol ␥ 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 ␥ has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol ␥ 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 ␥ 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 ␥.
Regulation and Modulation of Human DNA Polymerase δ Activity and Function
2017
This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, PDIP46 and PDIP38, both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and ...
Journal of Biological Chemistry, 2001
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 ␥ (pol ␥), we investigated the fidelity of DNA synthesis by human pol ␥. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol ␥ has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol ␥ 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 ␥ has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol ␥ 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 ␥ 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 ␥.
High fidelity and lesion bypass capability of human DNA polymerase δ
Biochimie, 2009
DNA polymerase δ (Pol δ) is one of the main replicative DNA polymerases in human cells and therefore is a critical determinant of the overall accuracy of DNA synthesis. Here we document the fidelity of a human Pol δ holoenzyme and systematically score the types of mutations that the enzyme generates in a forward mutation assay. We find that human Pol δ is highly accurate, catalyzing less than one nucleotide mis-insertion per 220,000 nucleotides polymerized. Inactivation of proofreading or mutation of a conserved active site residue significantly elevates the frequency of incorporation errors, demonstrating the contribution of both the base selection and proofreading domains to the overall accuracy of synthesis by Pol δ. The highly selective nature of the polymerase active site is also indicated by the stalling of Pol δ upon encountering multiple types of DNA lesions. However, DNA damage is not an absolute block to Pol δ progression. We propose that partial lesion bypass by Pol δ represents a balance between stalling to allow for repair of mutagenic lesions by specialized repair proteins and bypass of damage to allow for successful completion of DNA synthesis by Pol δ in the presence of weakly blocking DNA adducts.
The EMBO Journal, 1989
Communicated by F.Eckstein DNA polymerase-primase complex, isolated with an apparently undegraded a-subunit, was immunoaffinitypurified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the a-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage 4PX174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10-6 and 10 x 10-6. This value reflects the spontaneous mutation frequency of 4X174am16 phages in Escherichia coli, and is 10to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10-7 (1/2 880 000) for dGMP:Ttemp,,te mispairs, between 10-7 and 10-8 for dCMP:Ttempiate (1/35 000 000), dCMP:Atemplate (1/18 200 000) and dAMP:Gte.,a mispairs (1/16 500 000), and below 10-8 (1/100 000 000) for dTM[:Ttnpae, dGMP:Anpate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'-5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide. Upon replacing dGTP by its phosphorothioate analogue at equimolar concentrations of the four nucleoside triphosphates, a 2-fold increase in the number of revertants was observed; biasing dGTPaS 9-and 30-fold over dATP and dCTP, respectively, led to a 50-fold increase in the number of revertants. Taken together, these observations suggest that the 3'-5' exonuclease present in immunoaffinity purified human polymerase-primase proofreads nucleotide misinsertions during DNA synthesis. The exonuclease contributes at least one to two orders of magnitude to the high fidelity characteristics of the intact polymerase-primase complex.
PLoS ONE, 2012
Mammalian DNA polymerase d (Pol d), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol d complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol d holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent k cat) of the Pol d holoenzyme, core+p68, core+p12 and p125/ p50 core were 100, 109, 40, and 29. The corresponding apparent K d 's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol d interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol d to adopt flexible configurations with PCNA. The abilities of the Pol d complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12.
Nucleic Acids Research
During DNA replication, DNA lesions in lagging strand templates are initially encountered by DNA polymerase δ (pol δ) holoenzymes comprised of pol δ and the PCNA processivity sliding clamp. These encounters are thought to stall replication of an afflicted template before the lesion, activating DNA damage tolerance (DDT) pathways that replicate the lesion and adjacent DNA sequence, allowing pol δ to resume. However, qualitative studies observed that human pol δ can replicate various DNA lesions, albeit with unknown proficiencies, which raises issues regarding the role of DDT in replicating DNA lesions. To address these issues, we re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with DNA lesions. The results indicate pol δ holoenzymes support dNTP incorporation opposite and beyond multiple lesions and the extent of these activities depends on the lesion and pol δ proofreading. Furthermore, after encountering a given...
A Novel DNA Damage Response: RAPID DEGRADATION OF THE p12 SUBUNIT OF DNA POLYMERASE
Journal of Biological Chemistry, 2007
Mammalian DNA polymerase (Pol) ␦ is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol ␦ in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol ␦ by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol ␦ function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR ؊/؊ cells but not in ATM ؊/؊ cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol ␦ in both DNA replication and DNA repair. . 2 The abbreviations used are: Pol, DNA polymerase; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiectasia mutated and RAD3-related; RPA, replication protein A; RFC, replication factor C; PCNA, proliferating cell nuclear antigen; Chk1-pS345, Chk1-phosphoserine 345; MMS, methyl methanesulfonate; HU, hydroxyurea; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; DTT, dithiothreitol.
Nucleic Acids Research, 2008
Human DNA polymerase d (Pol d4), a key enzyme in chromosomal replication, is a heterotetramer composed of the p125, p50, p68 and p12 subunits. Genotoxic agents such as UV and alkylating chemicals trigger a DNA damage response in which Pol d4 is converted to a trimer (Pol d3) by degradation of p12. We show that Pol d3 has altered enzymatic properties: it is less able to perform translesion synthesis on templates containing base lesions (O 6-MeG, 8-oxoG, an abasic site or a thyminethymine dimer); a greater proofreading activity; an increased exonuclease/polymerase activity ratio; a decreased tendency for the insertion of wrong nucleotides, and for the extension of mismatched primers. Overall, our findings indicate that Pol d3 exhibits an enhanced ability for the detection of errors in both primers and templates over its parent enzyme. These alterations in Pol d3 show that p12 plays a major role in Pol d4 catalytic functions, and provides significant insights into the rationale for the conversion of Pol d4 to Pol d3 in the cellular response to DNA damage.