Role Of DNA Polymerase Research Articles

The role of DNA polymerase I in tolerating single-strand breaks generated at clustered DNA damage in Escherichia coli

Clustered DNA damage, when multiple lesions are generated in close proximity, has various biological consequences, including cell death, chromosome aberrations, and mutations. It is generally perceived as a hallmark of ionizing radiation. The enhanced mutagenic potential of lesions within a cluster has been suggested to result, at least in part, from the selection of the strand with the mutagenic lesion as the preferred template strand, and that this process is relevant to the tolerance of persistent single-strand breaks generated during an attempted repair. Using a plasmid-based assay in Escherichia coli, we examined how the strand bias is affected in mutant strains deficient in different DNA polymerase I activities. Our study revealed that the strand-displacement and 5′-flap endonuclease activities are required for this process, while 3′-to-5′ exonuclease activity is not. We also found the strand template that the mutagenic lesion was located on, whether lagging or leading, had no effect on this strand bias. Our results imply that an unknown pathway operates to repair/tolerate the single-strand break generated at a bi-stranded clustered damage site, and that there exist different backup pathways, depending on which DNA polymerase I activity is compromised.

Small tandem DNA duplications result from CST-guided Pol \u03b1-primase action at DNA break termini

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3′ ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3′ tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3′ overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.

DNA polymerase β may be involved in protecting human bronchial epithelial cells from the toxic effects induced by methyl tert-butyl ether exposure.

Methyl tert-butyl ether (MTBE), a widely used gasoline additive and a ubiquitous environmental pollutant in many countries and regions, can cause various kinds of toxic effects on human health. However, the molecular mechanism underlying its toxic effects remains elusive. The present study aimed to explore the cytotoxicity, DNA damage and oxidative damage effects of MTBE on human bronchial epithelial cells (16HBE) and the possible role of DNA polymerase β (pol-β) in this process. RNA interference (RNAi) was used to obtain pol-β gene knocked-down cells (pol-β-). CCK-8 assay was adopted to analyze the cell viability. Alkaline single-cell gel electrophoresis (SCGE) was performed to detect the DNA damage effects of MTBE. The enzyme activity of GSH-Px, SOD, CAT and the level of MDA were assessed. The data indicated that when treated with MTBE at the concentration exceeding 50 μmol/L and for the time exceeding 24 h, the pol-β- exhibited significantly decreased cell viability and increased DNA damage effects, as compared to the control (P < 0.05). Furthermore, there was significant difference in the levels of GSH-pX, SOD, CAT and MDA between the pol-β- and the control (P < 0.05). Our investigation suggests that MTBE can cause obvious cytotoxicity, DNA damage and oxidative damage effects on 16HBE cells. DNA polymerase β may be involved in protecting 16HBE cells from the toxic effects induced by MTBE exposure. These findings provide a novel insight into the molecular mechanism underlying the toxic effects of MTBE on human cells.

The role of DNA polymerase ζ in benzo[a]pyrene-induced mutagenesis in the mouse lung.

DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose-response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.

A novel role of DNA polymerase λ in translesion synthesis in conjunction with DNA polymerase ζ.

By extending synthesis opposite from a diverse array of DNA lesions, DNA polymerase (Pol) ζ performs a crucial role in translesion synthesis (TLS). In yeast and cancer cells, Rev1 functions as an indispensable scaffolding component of Polζ and it imposes highly error-prone TLS upon Polζ. However, for TLS that occurs during replication in normal human cells, Rev1 functions instead as a scaffolding component of Pols η, ι, and κ and Rev1-dependent TLS by these Pols operates in a predominantly error-free manner. The lack of Rev1 requirement for Polζ function in TLS in normal cells suggested that some other protein substitutes for this Rev1 role. Here, we identify a novel role of Polλ as an indispensable scaffolding component of Polζ. TLS studies opposite a number of DNA lesions support the conclusion that as an integral component, Polλ adapts Polζ-dependent TLS to operate in a predominantly error-free manner in human cells, essential for genome integrity and cellular homeostasis.

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.

SummaryAs a promising target for alternative antimicrobials, bacterial recombinase A (RecA) protein has attracted much attention for its roles in antibiotic-driven SOS response and mutagenesis. Naphthalene polysulfonated compounds (NPS) such as suramin have previously been explored as antibiotic adjuvants targeting RecA, although the underlying structural bases for RecA-ligand interactions remain obscure. Based on our in silico predictions and documented activity of NPS in vitro, we conclude that the analyzed NPS likely interact with Tyr103 (Y103) and other key residues in the ATPase activity center (pocket A). For validation, we generated recombinant RecA proteins (wild-type versus Y103 mutant) to determine the binding affinities for RecA protein interactions with suramin and underexamined NPS in isothermal titration calorimetry. The corresponding dissociation constants (Kd) ranged from 11.5 to 18.8 μM, and Y103 was experimentally shown to be critical to RecA-NPS interactions.

Roles for DNA polymerase \u03b4 in initiating and terminating leading strand DNA replication

Most current evidence indicates that DNA polymerases ε and δ, respectively, perform the bulk of leading and lagging strand replication of the eukaryotic nuclear genome. Given that ribonucleotide and mismatch incorporation rates by these replicases influence somatic and germline patterns of variation, it is important to understand the details and exceptions to this overall division of labor. Using an improved method to map where these replicases incorporate ribonucleotides during replication, here we present evidence that DNA polymerase δ universally participates in initiating leading strand synthesis and that nascent leading strand synthesis switches from Pol ε to Pol δ during replication termination. Ribonucleotide maps from both the budding and fission yeast reveal conservation of these processes. These observations of replisome dynamics provide important insight into the mechanisms of eukaryotic replication and genome maintenance.

Synthesis of N2 -Aryl-2'-Deoxyguanosine Modified Phosphoramidites and Oligonucleotides.

The N2 -position of 2'-deoxyguanosine (N2 -position in dG) is well known for forming carcinogenic minor groove DNA adducts, which originate from environmental pollutants, chemicals, and tobacco smoke. The N2 -dG DNA adducts have strong implications on biological processes such as DNA replication and repair and may, therefore, result in genomic instability by generating mutations or even cell death. It is crucial to know the role of DNA polymerases when they encounter the N2 -dG damaged site in DNA. To get detailed insights on the in vitro DNA damage tolerance or bypass mechanism, there is a need to synthetically access N2 -dG damaged DNAs. This article describes a detailed protocol of the synthesis of N2 -aryl-dG modified nucleotides using the Buchwald-Hartwig reaction as a main step and incorporation of the modified nucleotides into DNA. In Basic Protocol 1, we focused on the synthesis of five different N2 -dG modified phosphoramidites with varying bulkiness (benzyl to pyrenyl). Basic Protocol 2 describes the details of synthesizing N2 -dG modified oligonucleotides employing the standard solid phase synthesis protocol. This strategy provides robust synthetic access to various modifications at the N2 -position of dG; the modified dGs serve as good substrates to study translesion synthesis and repair pathways. Overall data presented in this article are based on earlier published reports. © 2019 by John Wiley & Sons, Inc.

Role of DNA Polymerase κ in the Processing of DNA-protein Cross-link Damage Induced by 2ʹ-deoxy-5-azacytidine and Formaldehyde

Genomic DNA that essential for cell survival is constantly undergoes various forms of DNA damages upon attacked by DNA-damaging agents from exogenous and endogenous sources. DNA-protein cross-links (DPCs) are super-bulky, steric hindrance and less characterized DNA damage among those so far identified. Currently known DPCs are classified into four main types depending on the way of attachment to DNA strands. Of these types, type 1 is the most ubiquities in which cross-linked proteins (CLPs) are covalently attached to an undistorted DNA strand. While several researchers worldwide start to be attention about DPC damage, the repair factors that are indispensable for the processing of type 1 DPC remain largely elusive. Therefore, in the present study, we analyzed the role of translesion synthesis (TLS) DNA polymerases κ and ι (polκ and ι) in the processing of type 1 DPC. Obviously, mouse cells deficient in polκ were highly sensitive to 2ʹ-deoxy-5-azacytidine (azadC, a DNA methylating agent) and formaldehyde (FA, a simple aldehyde). Furthermore, the quantitative analysis of DPCs in polκ proficient and deficient cells using fluorescence labeling method which we have developed recently revealed that the amount of DPCs increased significantly in azadC and FA-treated cells compared to untreated control. In contrast, a DNA methylation inhibitor Zebularine (Zeb) does not enhance the sensitivity of polκ deficient cells compared to polκ proficient cells. Additionally, no DPC is formed upon treatment with Zeb in polκ cells. The most remarkable conclusion is that the sensitivity of polκ deficient cells to azadC is exclusively due to DPC and ruling out the involvement of polκ in DNA methylation. Based on the current findings, we suggested a possible repair model for type 1 DPC induced by azadC and FA. Wherein, small peptides result from breakage of large CLPs are bypassed by polκ and consequently the repair proceeds.

The Role of DNA Polymerase β in Neural Genome Stability.

Development of the nervous system requires robust expansion of progenitor cells that will generate a diverse population of long-lived mature neurons. Precise transmission of the genomic information from parent cell to daughter cells and the faithful maintenance of this information in the terminally

Dual loss of human POLQ and LIG4 abolishes random integration

Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. Here we show that DNA polymerase θ, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase θ and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Thus, DNA polymerase θ-mediated end joining is the sole homology-independent repair route in the absence of NHEJ and, intriguingly, their combined absence reveals rare Alu-Alu recombination events utilizing a stretch of homology. Our findings provide new insights into the mechanics of foreign DNA integration and the role of DNA polymerase θ in human genome maintenance.

Role of DNA polymerase β oxidized nucleotide insertion in DNA ligation failure

Production of reactive oxygen and nitrogen species (ROS), such as hydrogen peroxide, superoxide and hydroxyl radicals, has been linked to cancer, and these oxidative molecules can damage DNA. Base excision repair (BER), a major repair system maintaining genome stability over a lifespan, has an important role in repairing oxidatively induced DNA damage. Failure of BER leads to toxic consequences in ROS-exposed cells, and ultimately can contribute to the pathobiology of disease. In our previous report, we demonstrated that oxidized nucleotide insertion by DNA polymerase β (pol β) impairs BER due to ligation failure and leads to formation of a cytotoxic repair intermediate. Biochemical and cytotoxic effects of ligation failure could mediate genome stability and influence cancer therapeutics. In this review, we discuss the importance of coordination between pol β and DNA ligase I during BER, and how this could be a fundamental mechanism underlying human diseases such as cancer and neurodegeneration. A summary of this work was presented in a symposium at the International Congress of Radiation Research 2015 in Kyoto, Japan.

The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD>KO>WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA.

DNA polymerases β and λ and their roles in cell.

Among the set of mammalian DNA polymerases, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are involved in repair, including base excision repair and non-homologous end joining. Some of them play a crucial role during the specific process which is referred to as translesion synthesis (TLS). TLS intends for the cell surviving during the replication of damaged DNA templates. Additionally, specific activities of TLS-polymerases have to be useful for repair of double-stranded clustered lesions: if the synthesis is proceeded via base excision repair process, the role of DNA polymerases β or λ will be important. In this review we discussed the biochemical properties and functional relevance of X family DNA polymerases β and λ.

A role for DNA polymerase θ in the timing of DNA replication.

Although DNA polymerase θ (Pol θ) is known to carry out translesion synthesis and has been implicated in DNA repair, its physiological function under normal growth conditions remains unclear. Here we present evidence that Pol θ plays a role in determining the timing of replication in human cells. We find that Pol θ binds to chromatin during early G1, interacts with the Orc2 and Orc4 components of the Origin recognition complex and that the association of Mcm proteins with chromatin is enhanced in G1 when Pol θ is downregulated. Pol θ-depleted cells exhibit a normal density of activated origins in S phase, but early-to-late and late-to-early shifts are observed at a number of replication domains. Pol θ overexpression, on the other hand, causes delayed replication. Our results therefore suggest that Pol θ functions during the earliest steps of DNA replication and influences the timing of replication initiation.

A Role for DNA Polymerase θ in Promoting Replication through Oxidative DNA Lesion, Thymine Glycol, in Human Cells

The biological functions of human DNA polymerase (pol) θ, an A family polymerase, have remained poorly defined. Here we identify a role of polθ in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (TG) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, polymerases κ and ζ function together and mediate error-free TLS, whereas in the other, polθ functions in an error-prone manner. Human polθ is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite TG in human cells. In contrast to TLS mediated by polκ and polζ, in which polζ would elongate the chain from the TG:A base pair formed by polκ action, the ability of polθ alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5' template base, suggests that the polθ active site can accommodate highly distorting DNA lesions.

DNA Polymerase as a Molecular Motor and Pump

DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid pumps using enzymes as engines.

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Mutagenic Bypass of 8-Oxo-7,8-dihydroguanine (8-Hydroxyguanine) by DNA Polymerase κ in Human Cells

The formation of 8-oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA and in the nucleotide pool results in G:C→T:A and A:T→C:G substitution mutations, respectively, since G(O) can pair with both C and A. In this study, the role of DNA polymerase κ in the mutagenicity of G(O) was investigated, using a supF shuttle plasmid propagated in human U2OS cells. This translesion synthesis DNA polymerase was knocked down by siRNA, and plasmid DNAs containing G(O):C and G(O):A pairs were transfected into the knock-down cells. The supF plasmid DNAs replicated in the cells were then introduced into Escherichia coli. Mutation analyses indicated that the knock-down of DNA polymerase κ by siRNA decreased the frequency of G:C→T:A mutation caused by G(O):C, although no effects of the DNA polymerase κ reduction were observed for the A:T→C:G substitution induced by G(O):A. These results suggested that DNA polymerase κ is involved in the mutagenic bypass of G(O) in living human cells, when the damaged base is generated by direct DNA oxidation.

DNA Polymerase δ and ζ Switch by Sharing Accessory Subunits of DNA Polymerase δ

Translesion DNA synthesis is an important branch of the DNA damage tolerance pathway that assures genomic integrity of living organisms. The mechanisms of DNA polymerase (Pol) switches during lesion bypass are not known. Here, we show that the C-terminal domain of the Pol ζ catalytic subunit interacts with accessory subunits of replicative DNA Pol δ. We also show that, unlike other members of the human B-family of DNA polymerases, the highly conserved and similar C-terminal domains of Pol δ and Pol ζ contain a [4Fe-4S] cluster coordinated by four cysteines. Amino acid changes in Pol ζ that prevent the assembly of the [4Fe-4S] cluster abrogate Pol ζ function in UV mutagenesis. On the basis of these data, we propose that Pol switches at replication-blocking lesions occur by the exchange of the Pol δ and Pol ζ catalytic subunits on a preassembled complex of accessory proteins retained on DNA during translesion DNA synthesis.