Short-patch Base Excision Repair Research Articles

The enzymatic properties of Arabidopsis thaliana DNA polymerase λ suggest a role in base excision repair

Base excision repair (BER) generates gapped DNA intermediates containing a 5′-terminal 2-deoxyribose-5-phosphate (5′-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of Arabidopsis thaliana Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5′-end of the gap and is not inhibited by the presence of a 5′-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol β homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.

Oxidative DNA damage on the VEGF G-quadruplex forming promoter is repaired via long-patch BER.

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased ~two-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol β performs multiple insertions on G4 substrates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol β products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol β prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

Human DNA ligases I and IIIα as determinants of accuracy and efficiency of base excision DNA repair

Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3′-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase β-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3′-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5′-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.

Active DNA demethylation promotes cell fate specification and the DNA damage response.

Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.

A novel polymerase β inhibitor from phage displayed peptide library augments the anti-tumour effects of temozolomide on colorectal cancer

The therapeutic efficacy of TMZ, a common used drug for chemotherapy, is limited by the resistance from colorectal cancer cells. Base excision repair (BER) pathway has been identified as one of the reasons for drug resistance. By blocking Polβ-dependent BER (Base Excision Repair) pathway, the efficacy of TMZ treatment can be improved greatly. Several Polβ inhibitors that have been identified could not become approved drugs due to lack of potency or specificity. To find therapeutic candidates with exquisite specificity and high affinity to Polβ, phage display technology was used in the current research. We screened out a candidate Polβ inhibitor, 10 D, that can inhibit the activity of Polβand SP-BER (Short-Patch Base excision Repair) pathway. Co-treatment with 10 D enhanced the sensitivity of colorectal cancer (CRC) cells to TMZ both in vitro and in vivo. Our data suggested that the novel Polβ inhibitor we identified can improve TMZ efficacy and optimize CRC chemotherapy.

Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage.

On induction of DNA damage with 405-nm laser light, proteins involved in base excision repair (BER) are recruited to DNA lesions. We find that the dynamics of factors typical of either short-patch (XRCC1) or long-patch (PCNA) BER are altered by chemicals that perturb actin or tubulin polymerization in human cells. Whereas the destabilization of actin filaments by latrunculin B, cytochalasin B, or Jasplakinolide decreases BER factor accumulation at laser-induced damage, inhibition of tubulin polymerization by nocodazole increases it. We detect no recruitment of actin to sites of laser-induced DNA damage, yet the depolymerization of cytoplasmic actin filaments elevates both actin and tubulin signals in the nucleus. While published evidence suggested a positive role for F-actin in double-strand break repair in mammals, the enrichment of actin in budding yeast nuclei interferes with BER, augmenting sensitivity to Zeocin. Our quantitative imaging results suggest that the depolymerization of cytoplasmic actin may compromise BER efficiency in mammals not only due to elevated levels of nuclear actin but also of tubulin, linking cytoskeletal integrity to BER.

Abstract 2078: A multiscale map of DNA damage response in human cells

Abstract The DNA damage response (DDR) ensures error-free genome replication and transcription, but is often disrupted in cancer. We developed DDRAM, a multilayered map of DDR derived from our own high-throughput physical interaction screen as well as genome, transcriptome, and proteome datasets from published sources. DDRAM organizes 328 genes into 41 hierarchical pathways. We identify 55 (17%) novel DDR genes with functions distributed across various pathways, and novel and more specific roles for 153 (47%) of established DDR genes. We perform epistasis mapping to support novel pathway assignments of FOXK1 in nucleotide metabolism and XRCC3 in the Fanconi anemia pathway. Using quantitative imaging of a fluorescent probe, in some cases confirmed by epistasis mapping, we experimentally validate members of the Short Patch Base Excision Repair pathway over a time course, revealing a more complete model of poly(ADP-ribose)-dependent recruitment to sites of DNA damage. Citation Format: Anton Kratz, Minkyu Kim, Fan Zheng, Christopher A. Koczor, Samson Fong, Jianfeng Li, Robert W. Sobol, Nevan Krogan, Trey Ideker. A multiscale map of DNA damage response in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2078.

Participation of RecJ in the base excision repair pathway of Deinococcus radiodurans.

RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ) deletion strain exhibited extreme sensitivity to hydrogen peroxide and methyl-methanesulphonate, as well as a high spontaneous mutation rate and an accumulation of unrepaired abasic sites in vivo, indicating the involvement of drRecJ in the BER pathway. The binding affinity and nuclease activity preference of drRecJ toward DNA substrates containing a 5′-P-dSpacer group, a 5′-deoxyribose-phosphate (dRP) mimic, were established. A 1.9 Å structure of drRecJ in complex with 5′-P-dSpacer-modified single-stranded DNA (ssDNA) revealed a 5′-monophosphate binding pocket and occupancy of 5′-dRP in the drRecJ nuclease core. The mechanism for RecJ 5′-dRP catalysis was explored using structural and biochemical data, and the results implied that drRecJ is not a canonical 5′-dRP lyase. Furthermore, in vitro reconstitution assays indicated that drRecJ tends to participate in the long-patch BER pathway rather than the short-patch BER pathway.

Effect of Human XRCC1 Protein Oxidation on the Functional Activity of Its Complexes with the Key Enzymes of DNA Base Excision Repair

Base excision repair (BER) ensures correction of most abundant DNA lesions in mammals. The efficiency of this multistep DNA repair process that can occur via different pathways depends on the coordinated action of enzymes catalyzing its individual steps. The scaffold XRCC1 (X-ray repair cross-complementing protein 1) protein plays an important coordinating role in the repair of damaged bases and apurinic/apyrimidinic (AP) sites via short-patch (SP) BER pathway, as well as in the repair of single-strand DNA breaks. In this study, we demonstrated for the first time in vitro formation of the ternary XRCC1 complex with the key enzymes of SP BER - DNA polymerase β (Polβ) and DNA ligase IIIα (LigIIIα) - using the fluorescence-based technique. It was found that Polβ directly interacts with LigIIIα, but their complex is less stable than the XRCC1-Polβ and XRCC1-LigIIIα complexes. The effect of XRCC1 oxidation and composition of the multiprotein complex on the efficiency of DNA synthesis and DNA ligation during DNA repair has been explored. We found that formation of the disulfide bond between Cys12 and Cys20 residues as a result of XRCC1 oxidation (previously shown to modulate the protein affinity for Polβ), affects the yield of the final product of SP BER and of non-ligated DNA intermediates (substrates of long-patch BER). The effect of XRCC1 oxidation on the final product yield depended on the presence of AP endonuclease 1. Together with the data from our previous work, the results of this study suggest an important role of XRCC1 oxidation in the fine regulation of formation of BER complexes and their functional activity.

PARP inhibitor Olaparib Enhances the Apoptotic Potentiality of Curcumin by Increasing the DNA Damage in Oral Cancer Cells through Inhibition of BER Cascade.

Although Olaparib (Ola, a PARP-inhibitor), in combination with other chemotherapeutic agents, was clinically approved to treat prostate cancer, but cytotoxicity, off-target effects of DNA damaging agents limit its applications in clinic. To improve the anti-cancer activity and to study the detailed mechanism of anti-cancer action, here we have used bioactive compound curcumin (Cur) in combination with Ola. Incubation of Ola in Cur pre-treated cells synergistically increased the death of oral cancer cells at much lower concentrations than individual optimum dose and inhibited the topoisomerase activity. Short exposure of Cur caused DNA damage in cells, but more increased DNA damage was noticed when Ola has incubated in Cur pre-treated cells. This combination did not alter the major components of homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways but significantly altered both short patch (SP) and long patch (LP) base excision repair (BER) components in cancer cells. Significant reduction in relative luciferase activity, expression of BER components and PARylation after Cur and Ola treatment confirmed this combination inhibit the BER activity in cells. Reduction of PARylation, decreased expression of BER components, decreased tumor volume and induction of apoptosis were also noticed in Cur + Ola treated Xenograft mice model. The combination treatment of Cur and Ola also helped in recovering the body weight of tumor-bearing mice. Thus, Cur + Ola combination increased the oral cancer cells death by not only causing the DNA damage but also blocking the induction of BER activity.

A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates

Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD+ as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.

Nanoquinacrine sensitizes 5-FU-resistant cervical cancer stem-like cells by down-regulating Nectin-4 via ADAM-17 mediated NOTCH deregulation.

Cervical cancer is a major cause of cancer-related death in women world-wide. Although the anti-metabolite 5-FU is widely used for its treatment, its clinical utility is limited due to the frequent occurrence of drug resistance during metastasis. Cancer stem-like cells (CSCs), present in the heterogeneous population of CC cells, are thought to contribute to this resistance. Nectin-4, a CSC marker, is known to play an important role in the cellular aggressiveness associated with metastatic CC. This study was designed to assess the role of Nectin-4 in the acquisition of 5-FU resistance by metastatic CC cells, including its relation to the NOTCH signalling pathway. 5FU-resistant CC cell lines were deduced from ME-180 and SiHA cells by continuous exposure to a single concentration of 5-FU. Thymidylate synthase (TS) positive cells were isolated from the 5-FU resistant cells, after which a metastatic model was developed. The role of Nectin-4 in the sensitization of 5-FU resistant metastatic CC cells upon incubation with Nano-formulated Quinacrine (NQC) was investigated using multiple bioassays including MTT, FACS, ELISA, immunoflurescence, Western blotting, comet and in vivo plasmid-based short patch and long patch base excision repair assays. We found that the expression level of Nectin-4, as well as that of other CSC markers (Oct-4, β-catenin, SOX2) and representative NOTCH signalling components (NOTCH-1, Jagged-1, γ-secretase, ADAM-17) were elevated in the 5-FU resistant metastatic cells compared to those in control cells. Increased nuclear translocation of Nectin-4 and increased proliferation and invasion rates were observed after culturing the metastatic cells under hypoxic conditions. Treatment with NQC inhibited the nuclear translocation of Nectin-4 and decreased the proliferation and invasion rates of the cells by inhibiting the induction of base excision repair (BER) pathway components and ADAM-17 expression levels. After combination treatment of Nectin-4 overexpressing metastatic CC cells with a specific ADAM-17 inhibitor (GW280264) and NQC, a decreased Nectin-4 expression, without alterations in BER and/or other NOTCH pathway components, was noted. Our data indicate that Nectin-4 may play a prominent role in 5-FU resistance of metastatic CC cells and that NQC sensitizes these cells by Nectin-4 deregulation through ADAM-17 inhibition, a major component of the NOTCH signalling pathway.

The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase β

DNA polymerase β (Pol β) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol β incorporates a nucleotide into a single-gapped DNA substrate. Pol β may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5'-modified downstream DNA. We have recently shown that Pol β fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with single-nucleotide gapped DNA show that Pol β undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate. Here we use FRET to investigate nucleotide incorporation of Pol β with various DNA substrates. The results show that increasing the gap size influences the fingers closing step by increasing its reverse rate. However, the 5'-phosphate group has a more significant effect. The absence of the 5'-phosphate decreases the DNA binding affinity of Pol β and results in a conformationally more open binary complex. Moreover, upon addition of the correct nucleotide in the absence of 5'-phosphate, a slow fingers closing step is observed. Interestingly, either increasing the gap size or removing the 5'-phosphate group results in loss of the noncovalent step. Together, these results suggest that the character of the DNA substrate impacts the nature and rates of pre-catalytic conformational changes of Pol β. Our results also indicate that conformational changes are important for the fidelity of DNA synthesis by Pol β.

Identification of a unique insertion in plant organellar DNA polymerases responsible for 5′-dRP lyase and strand-displacement activities: Implications for Base Excision Repair

Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5′-deoxyribose phosphate (5′-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5′-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5′-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5′-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs.

Time-Dependent Extension from an 8-Oxoguanine Lesion by Human DNA Polymerase Beta

The oxidative DNA lesion 7,8-dihydro-2'-deoxyguanine (8-oxoG) often occurs in double-stranded DNA and poses a threat to genomic integrity due to the ability of 8-oxoG to form stable Watson-Crick base pairs with deoxycytidine (8-oxoG:dC) and Hoogsteen base pairs with deoxyadenosine (8-oxoG:dA). In humans, short-patch base excision repair of 8-oxoG:dA base pairs requires human DNA polymerase β (hPolβ) to bypass 8-oxoG. Previously, we have shown hPolβ-catalyzed 8-oxoG bypass to exhibit low fidelity and identified a unique stacking interaction between the newly incorporated nucleotide (dCMP or dAMP) and the templating 8-oxoG. The effect of this stacking on the ability of hPolβ to extend from 8-oxoG during long-patch base excision repair was unknown. Here we report pre-steady-state kinetics and time-dependent crystal structures to demonstrate that extension from both 8-oxoG:dC and 8-oxoG:dA base pairs is 18- to 580-fold less efficient compared to 8-oxoG bypass and that extension from 8-oxoG:dC over 8-oxoG:dA is favored by 15-fold. The overall decrease in efficiency of extension relative to 8-oxoG bypass is due to an alternative nucleotide binding conformation in the precatalytic ternary structures (hPolβ·DNA·dNTP) for both extension contexts, wherein the incoming nucleotide is bound in either the canonical Watson-Crick base pair or a nonplanar base pair. In addition, the decreased stability of the ternary complex of 8-oxoG:dA extension results in further loss of efficiency when compared to 8-oxoG:dC extension. Therefore, we hypothesize that the inefficient extension from 8-oxoG:dA serves as a newly discovered fidelity checkpoint during base excision repair.

Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation.

Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in vivo in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes. Using time-lapse analysis of cells expressing fluorescently tagged repair proteins and also validation of the DNA damage generated by micro-irradiation using several key damage markers, we show that irradiation with a 405 nm continuous wave laser lead to the activation of all repair pathways even in the absence of exogenous sensitization. In contrast, we found that irradiation with 488 nm laser lead to the selective activation of non-processive short-patch base excision and single strand break repair, which were further validated by PARP inhibition and metoxyamine treatment. We conclude that these low energy conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.

The Human Ligase IIIα-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin.

Overexpression of A613T and G462T variants of DNA polymerase β weakens chemotherapy sensitivity in esophageal cancer cell lines.

BackgroundHuman DNA polymerase β (polβ) is a small monomeric protein that is essential for short-patch base excision repair. It plays an important role in regulating the sensitivity of tumor cells to chemotherapy.MethodsWe evaluated the mutation of polβ in a larger cohort of esophageal cancer (EC) patients by RT-PCR and sequencing analysis. The function of the mutation was evaluated by CCK-8, in vivo tumor growth, and flow cytometry assays.ResultsThere are 229 patients with the polβ mutation, 18 patients with A613T mutation, 12 patients with G462T mutation among 538 ECs. Analysis results of survival time showed that EC patients with A613T, G462T mutation had a shorter survival than the others (P < 0.05). CCK-8 and flow cytometry assays results showed the A613T, G462T EC9706 cells were less sensitive than WT cells to 5-FU and cisplatin (P < 0.05). Experiments results in vivo showed that the tumor sizes of A613T and G462T group were larger than WT and polβ−/− groups (P < 0.05).ConclusionsIn this study, we discovered A to T point mutation at nucleotide 613 (A613T) and G to T point mutation at nucleotide 462 (G462T) in the polβ gene through 538 EC patients cohort study. A613T and G462T variant of DNA polymerase β weaken chemotherapy sensitivity of esophageal cancer.

Strand displacement synthesis by yeast DNA polymerase ε.

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair.

Activity of DNA polymerase λ in spikelets of rice and maize

Involvement of DNA polymerase (pol) enzymes in meiotic DNA repair has not been clearly understood in plants. DNA polymerase λ is involved in short patch DNA synthesis and base excision repair in both plants and animals. The presence and activity of the pol λ enzyme was studied in a protein isolated from spikelets during flower development stages of rice (Oryza sativa L.) and maize (Zea mays L.). Western blot analysis showed a 2- to 2.5-fold higher accumulation of pol λ in spikelets than in shoots. Assays of pol and in-gel activity showed the dideoxynucleotide triphosphate sensitive pol λ enzyme in spikelets of both the plants. An enhanced presence of the enzyme and its high activity suggests an active role of pol λ in meiotic recombination during microspore development.