Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).     

Cooperation of terminal deoxynucleotidyl transferase with DNA polymerase α in the replication of ultraviolet-irradiated DNA

The amount of DNA synthesis in vitro with the ultraviolet-irradiated poly-(dT) · oligo(rA) template initiators catalysed by DNA polymerase α (Masaki, S. and Yoshida, S., Biochim. Biophys. Acta 521, 74–88) decreased with the dose of ultraviolet-irradiation. The ultraviolet irradiation to the template, however, did not affect the rate of incorporation of incorrect deoxynycleotides into the newly synthesized poly(dA). The addition of terminal deoxynucleotidyl transferase to this system enhanced the DNA synthesis to a level which is comparable to that of the control and it concomitantly increased the incorporation of the mismatched deoxynucleotide into the newly synthesized poly(dA) strands. On the other hand, with an unirradiated template initiator, the misincorporation was only slightly enhanced by the addition of terminal deoxynucleotidyl transferase. The sizes of newly synthesized DNA measured by the sedimentation velocities were found to be smaller with the ultraviolet-irradiated templates but they increased to the control level with the addition of terminal deoxynucleotidyl transferase to the systems. These results suggest that terminal deoxynucleotidyl transferase can help DNA polymerase α to ‘bypass’ thymine dimers in vitro by the formation of mismatched regions at the positions opposite to pyrimidine dimers on the template.     

Structure and mechanism of error-free replication past the major benzo[a]pyrene adduct by human DNA polymerase κ

Benzo[a]pyrene (BP) is a well-known and frequently encountered carcinogen which generates a bulky DNA adduct (+)-trans-10S-BP-N²-dG (BP-dG) in cells. DNA polymerase kappa (polκ) is the only known Y-family polymerase that bypasses BP-dG accurately and thus protects cells from genotoxic BP. Here, we report the structures of human polκ in complex with DNA containing either a normal guanine (G) base or a BP-dG adduct at the active site and a correct deoxycytidine. The structures and supporting biochemical data reveal a unique mechanism for accurate replication by translesion synthesis past the major bulky adduct. The active site of polκ opens at the minor groove side of the DNA substrate to accommodate the bulky BP-dG that is attached there. More importantly, polκ stabilizes the lesion DNA substrate in the same active conformation as for regular B-form DNA substrates and the bulky BPDE ring in a 5′ end pointing conformation. The BP-dG adducted DNA substrate maintains a Watson–Crick (BP-dG:dC) base pair within the active site, governing correct nucleotide insertion opposite the bulky adduct. In addition, polκ''s unique N-clasp domain supports the open conformation of the enzyme and the extended conformation of the single-stranded template to allow bypass of the bulky lesion. This work illustrates the first molecular mechanism for how a bulky major adduct is replicated accurately without strand misalignment and mis-insertion.     

Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase η

N⁶-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication remain elusive. In this work, we used Y-family human DNA polymerase η as a model to investigate the kinetics of bypass of 6mA by hPol η. We found 6mA and its intermediate hypoxanthine (I) on template partially inhibited DNA replication by hPol η. dTMP incorporation opposite 6mA and dCMP incorporation opposite I can be considered as correct incorporation. However, both 6mA and I reduced correct incorporation efficiency, next-base extension efficiency, and the priority in extension beyond correct base pair. Both dTMP incorporation opposite 6mA and dCTP opposite I showed fast burst phases. However, 6mA and I reduced the burst incorporation rates (k_pol) and increased the dissociation constant (K_d,dNTP), compared with that of dTMP incorporation opposite unmodified A. Biophysical binding assays revealed that both 6mA and I on template reduced the binding affinity of hPol η to DNA in binary or ternary complex compared with unmodified A. All the results explain the inhibition effects of 6mA and I on DNA replication by hPol η, providing new insight in the effects of epigenetically modified 6mA on human DNA replication.     

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polɛ heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of the human Polɛ heterotetramer (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolɛ is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolɛ and sway hPolɛ toward DNA synthesis rather than proofreading.     

Study of the inhibitory effect of fatty acids on the interaction between DNA and polymerase β

The binding of human DNA polymerase β (pol β) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol β inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol β inhibitors. In the interaction between pol β and DNA, pol β could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol β inhibitors prevented the binding of pol β to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol β to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol β-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol β to DNA was mainly due to 7-fold increase in the dissociation rate. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 1000–1006. These authors contributed equally.     

Cell cycle control of β-cell replication in the prenatal and postnatal human pancreas

β-Cell regeneration declines with aging, but the molecular mechanisms controlling β-cell replication in humans are not well understood. We compared the expression of selected cell cycle proteins in prenatal and adult tissue and examined the association of these proteins with β-cell replication. Pancreatic tissue from a total of 20 human fetuses and adults was stained for Ki67, cyclin D3, p16 and p27, and insulin. The β-cellular expression of these cell cycle proteins was determined. The frequency of β-cell replication was lower in adult compared with prenatal β-cells (<0.5 vs. 3.4 ± 0.5%, respectively; P < 0.0001). p16 was sporadically expressed in prenatal β-cells (8.0 ± 1.1%) but highly enriched in adult β-cells (63.1 ± 5.2%, P < 0.0001). Likewise, the expression of p27 was much lower in prenatal β-cells (1.7 ± 0.4 vs. 44.1 ± 5.4%, respectively, P < 0.0001), and cyclin D3 expression increased from 24.2 ± 4.1 to 47.25 ± 5.0%, respectively (P < 0.001), with aging. The expression of all three proteins was significantly correlated with each other (P < 0.01 and r > 0.75, respectively). The strong expression of cyclin D3 in adult human β-cells and its correlation to p27 and p16 suggest a positive role in human β-cell cycle regulation. p16 and p27 appear to restrict β-cell replication with aging. The age dependency of cell cycle regulation in human β-cells might explain the reduced β-cell regeneration in adult humans.     

DNA polymerase ι: The long and the short of it!

The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein. However, the existence of the 740 amino acid polι has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of polι, we were unable to detect the longer 740 amino acid (ι-long) isoform in western blots. However, trace amounts of the ι-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid polι. We therefore purified and characterized recombinant full-length (740 amino acid) polι-long and compared it to full-length (715 amino acid) polι-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between ι-long and ι-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like ι-short, the ι-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of DNA polι in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform.     

Wheat DNA polymerase CI: a homologue of rat DNA polymerase β

We have previously described a low-molecular-weight DNA polymerase (52 kDa) from wheat embryo: DNA polymerase CI (pol CI). This enzyme shares some biochemical properties with animal DNA polymerase (pol ). In this report, we analyse pol CI in wheat embryo germination. Immunodetection and measurement of the enzyme activity show that wheat pol CI remains at a constant level during germination, whereas dramatic changes of the replicative DNA polymerase A and B activities were previously reported. We observe that the level of pol CI in physiological conditions (embryo germination and dividing cell culture) is in agreement with a pol -type DNA polymerase. By microsequencing of the electroblotted 52 kDa polypeptide, we determined the sequence of a dodecapeptide from the N-terminal region. A comparative analysis of the N-terminal pol CI peptide with some mammalian pol sequences shows a clear homology with helix 1 of the N-terminal ssDNA domain (residues 15 to 26) of the rat pol . Thus, the helical structure of this region should be conserved in the wheat peptide. This represents the first evidence of a partial primary structure of a -type DNA polymerase in plants.     

The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.     

The accessory subunit of mitochondrial DNA polymerase γ determines the DNA content of mitochondrial nucleoids in human cultured cells

The accessory subunit of mitochondrial DNA polymerase γ, POLGβ, functions as a processivity factor in vitro. Here we show POLGβ has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGβ increased nucleoid numbers, whereas over-expression of POLGβ reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGβ altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGβ preferentially bound to plasmids with a short displacement-loop, in contrast to POLGα. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGβ is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.     

Polymorphisms in the human DNA polymerase β gene

Recently, evidence has accumulated that mutations in DNA repair genes might be associated with certain steps in carcinogenesis. The DNA polymerase gene is one of the DNA repair genes, and mutations in it have been detected in 83% of human colorectal cancers. To assess the involvement of polymerase gene mutations in the development of human prostate cancers, we performed sequence analyses of human DNA samples. Unexpectedly, we found six regions that were polymorphic. This information should be taken into consideration at the time of sequence analysis of the DNA polymerase gene.s     

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.     

Defect of Dpb2p, a noncatalytic subunit of DNA polymerase ɛ, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae

The biological consequences of clusters containing a single strand break and base lesion(s) remain largely unknown. In the present study we determined the mutagenicities of two- and three-lesion clustered damage sites containing a 1-nucleotide gap (GAP) and 8-oxo-7,8-dihydroguanine(s) (8-oxoG(s)) in Escherichia coli. The mutation frequencies (MFs) of bi-stranded two-lesion clusters (GAP/8-oxoG), especially in mutY-deficient strains, were high and were similar to those for bi-stranded clusters with 8-oxoG and base lesions/AP sites, suggesting that the GAP is processed with an efficiency similar to the efficiency of processing a base lesion or an AP site within a cluster. The MFs of tandem two-lesion clusters comprised of a GAP and an 8-oxoG on the same strand were comparable to or less than the MF of a single 8-oxoG. The mutagenic potential of three-lesion clusters, which were comprised of a tandem lesion (a GAP and an 8-oxoG) and an opposing single 8-oxoG, was higher than that of a single 8-oxoG, but was no more than that of a bi-stranded 8-oxoGs. We suggest that incorporation of a nucleotide opposite 8-oxoG is less mutagenic when a GAP is present in a cluster than when a GAP is absent. Our observations indicate that the repair of a GAP is retarded by an opposing 8-oxoG, but not by a tandem 8-oxoG, and that the extent of GAP repair determines the biological consequences.     

The C-terminal domain of human Rev1 contains independent binding sites for DNA polymerase η and Rev7 subunit of polymerase ζ

Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (pol?). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the ’inserter’ polη and Rev7 subunit of the ’extender’ pol?, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS.

Structured summary of protein interactions

REV1, REV3 and REV7physically interact by nuclear magnetic resonance (View interaction), molecular sieving (View interaction) and isothermal titration calorimetry (View interaction).REV3 and REV7bind by molecular sieving (View interaction)REV1 and Polη-RIR peptidebind by nuclear magnetic resonance (View interaction)REV1, REV3, REV7 and Polη-RIR peptidephysically interact by nuclear magnetic resonance (View interaction).     

Interaction with DNA polymerase η is required for nuclear accumulation of REV1 and suppression of spontaneous mutations in human cells

Defects in the gene encoding human Polη result in xeroderma pigmentosum variant (XP-V), an inherited cancer-prone syndrome. Polη catalyzes efficient and accurate translesion DNA synthesis (TLS) past UV-induced lesions. In addition to Polη, human cells have multiple TLS polymerases such as Polι, Polκ, Polζ and REV1. REV1 physically interacts with other TLS polymerases, but the physiological relevance of the interaction remains unclear. Here we developed an antibody that detects the endogenous REV1 protein and found that human cells contain about 60,000 of REV1 molecules per cell as well as Polη. In un-irradiated cells, formation of nuclear foci by ectopically expressed REV1 was enhanced by the co-expression of Polη. Importantly, the endogenous REV1 protein accumulated at the UV-irradiated areas of nuclei in Polη-expressing cells but not in Polη-deficient XP-V cells. UV-irradiation induced nuclear foci of REV1 and Polη proteins in both S-phase and G1 cells, suggesting that these proteins may function both during and outside S phase. We reconstituted XP-V cells with wild-type Polη or with Polη mutants harboring substitutions in phenylalanine residues critical for interaction with REV1. The REV1-interaction-deficient Polη mutant failed to promote REV1 accumulation at sites of UV-irradiation, yet (similar to wild-type Polη) corrected the UV sensitivity of XP-V cells and suppressed UV-induced mutations. Interestingly however, spontaneous mutations of XP-V cells were only partially suppressed by the REV1-interaction deficient mutant of Polη. Thus, Polη–REV1 interactions prevent spontaneous mutations, probably by promoting accurate TLS past endogenous DNA lesions, while the interaction is dispensable for accurate Polη-mediated TLS of UV-induced lesions.     

Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β

The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.