Damaged DNA Strand Research Articles

Systematic analysis of natural topoisomerase I inhibitors from Forsythiae Fructus by ultrafiltration-UPLC-ESI-MS/MS, pharmacophore modelling, and molecular dynamics simulation

This study conducted a systematic analysis to explore natural DNA topoisomerase I (topo I) inhibitors from Forsythiae Fructus (FF). Crude extract of FF exhibited notable toxic and anti-proliferative effects on A549 cells. A total of 36 components were identified using bioaffinity ultrafiltration UPLC-ESI-MS/MS. Pinoresinol, 1,8-dihydrox­yanthraquinone, quercetin, and lariciresinol were screened as topo I inhibitors. Their ESI fragmentation patterns were analysed. An obvious repair effect on damaged DNA strands was observed by topo I inhibitory binding assay. Moreover, a common feature-based pharmacophore model was constructed and another 7 topo I inhibitors were screened. Molecular docking indicated that hydrogen bond, π-anion, and π-alkyl interaction were major interactions. Molecular dynamics simulation revealed important residues determining the binding of amentoflavone, forsythoside B and topo I. The results improved current understanding of natural topo I inhibitors from FF. Moreover, the combination of multi-disciplinary approaches provided a new tool to investigate natural antitumor products.

DNA lesion bypass and the stochastic dynamics of transcription-coupled repair

DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the subsequent triggering of repair processes have major roles in shaping the genome-wide distribution of mutations, clearing barriers to transcription, and minimizing the production of miscoded gene products. Despite its importance for genetic integrity, key mechanistic features of this transcription-coupled repair (TCR) process are controversial or unknown. Here, we exploited a well-powered in vivo mammalian model system to explore the mechanistic properties and parameters of TCR for alkylation damage at fine spatial resolution and with discrimination of the damaged DNA strand. For rigorous interpretation, a generalizable mathematical model of DNA damage and TCR was developed. Fitting experimental data to the model and simulation revealed that RNA polymerases frequently bypass lesions without triggering repair, indicating that small alkylation adducts are unlikely to be an efficient barrier to gene expression. Following a burst of damage, the efficiency of transcription-coupled repair gradually decays through gene bodies with implications for the occurrence and accurate inference of driver mutations in cancer. The reinitation of transcription from the repair site is not a general feature of transcription-coupled repair, and the observed data is consistent with reinitiation never taking place. Collectively, these results reveal how the directional but stochastic activity of TCR shapes the distribution of mutations following DNA damage.

Sperm DNA Fragmentation: Causes, Effects and Relevance in IVF Outcomes

Sperm DNA fragmentation, characterized by damaged or broken DNA strands within sperm cells, has been recognized as an important factor in male infertility and poor ART outcome. This lecture aims to provide an overview of the causes, effects, and relevance of sperm DNA fragmentation in in vitro fertilization (IVF) outcomes and look into the latest evidences from the literatures. Causes: Sperm DNA fragmentation can result from various factors, including oxidative stress, advanced paternal age, lifestyle factors such as smoking and excessive alcohol consumption, environmental exposures, genotoxic agents, and underlying genetic abnormalities. These factors can induce DNA damage during sperm development, during the transit of sperm through the male reproductive tract or during the storage of the sperm in epididymis. Relevance in IVF Outcomes: The assessment of sperm DNA fragmentation has emerged as a valuable tool in evaluating male infertility and predicting IVF success. Several methods, including the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL), comet assay, and other commercial detection kits, are available to measure sperm DNA fragmentation. Previous small cohort studies have demonstrated that higher levels of sperm DNA fragmentation are associated with lower pregnancy rates, decreased embryo quality, and an increased risk of pregnancy loss in IVF cycles. Therefore, incorporating sperm DNA fragmentation analysis into the routine assessment of male infertility can aid in optimizing treatment strategies and improving IVF outcomes. Recent evidences: Latest large clinical studies showed a limited association to IVF outcome. The DFI remains controversial as a predictive tool. Nonetheless, sperm DNA fragmentation is still believed to be a significant factor affecting male fertility and IVF outcomes. Further research is warranted to explore the mechanisms underlying DNA fragmentation, develop standardized assessment methods, and explore potential interventions to mitigate the adverse effects of sperm DNA damage on IVF success.

Sperm Chromatin Status and DNA Fragmentation in Mouse Species with Divergent Mating Systems.

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus, and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

Differing structures and dynamics of two photolesions portray verification differences by the human XPD helicase.

Ultraviolet light generates cyclobutane pyrimidine dimer (CPD) and pyrimidine 6-4 pyrimidone (6-4PP) photoproducts that cause skin malignancies if not repaired by nucleotide excision repair (NER). While the faster repair of the more distorting 6-4PPs is attributed mainly to more efficient recognition by XPC, the XPD lesion verification helicase may play a role, as it directly scans the damaged DNA strand. With extensive molecular dynamics simulations of XPD-bound single-strand DNA containing each lesion outside the entry pore of XPD, we elucidate strikingly different verification processes for these two lesions that have very different topologies. The open book-like CPD thymines are sterically blocked from pore entry and preferably entrapped by sensors that are outside the pore; however, the near-perpendicular 6-4PP thymines can enter, accompanied by a displacement of the Arch domain toward the lesion, which is thereby tightly accommodated within the pore. This trapped 6-4PP may inhibit XPD helicase activity to foster lesion verification by locking the Arch to other domains. Furthermore, the movement of the Arch domain, only in the case of 6-4PP, may trigger signaling to the XPG nuclease for subsequent lesion incision by fostering direct contact between the Arch domain and XPG, and thereby facilitating repair of 6-4PP.

Inhibiting and Enhancing Effects on DNA Repair of Rare Metal Elements in Cultured Human Lymphoblastoid Cells

Along with increasing relevance of rare earth (RE) elements in industrial technology, the risk of their environmental release and occupational exposure on human health is of concern. Although many toxicological studies were reported for REs, it is not known how they affect DNA repair. In this study, the effects on DNA repair of all RE ions except radioactive promethium (Pm) were studied. Human lymphoblastoid WTK1cells were irradiated to UV followed by 2h exposure to each RE with and without DNA repair inhibitor cytosine-1β-D- arabinofuranoside (araC), and then single strand breaks (SSBs) were detected by the comet assay. UV-induced pyrimidine dimers are removed by nucleotide excision repair (NER) which consists of recognition of the DNA lesion, excision of a 24–32 nucleotide stretch containing the lesion by dual incision of the damaged DNA strand on both sides, and re-synthesis of the resulting gap by DNA polymerase, and ligation of the nick. SSBs are generated in the incision step of nucleotide excision repair (NER) and disappear in the re-synthesis step of NER. Seven REs, Yb, Lu, Dy, Er, Sc, Pr, and Ce, enhanced comet positive response without araC but not with araC, suggesting that araC is antagonistic to the 7 REs. Since araC inhibits re-synthesis of NER, these seven REs would inhibit the re-synthesis step of NER. Six REs, Tm, Sm, Tb, Gd, Eu, and Y suppressed comet positive responses with and without araC, suggesting that they decreased comet assay detectable SSBs. Therefore, these 6 REs are considered to inhibit the incision step of NER. Only La decreased tail length without araC but increased with araC, suggesting that La increased comet assay detectable SSBs and that only La would enhance the incision step of NER. Neither Nd nor Ho affected tail length with or without araC.

LOCAL HYPERTHERMIA IN THE TREATMNET OF LOCALLY ADVANCED CERVICAL CANCER: CURRENT VIEW ON THE PROBLEM

The purpose of the study was to review available data on the combined use of local hyperthermia and chemotherapy/radiotherapy in the treatment of locally advanced cervical cancer, as well as to analyze longterm treatment outcomes.Material and methods. A systemic literature review was conducted using medline, cochrane library, and elibrary databases in the interval time between 2003 and 2020.Results. The review describes the mechanisms of biological efficiency of local hyperthermia and evaluates the effect of hyperthermia combined with chemotherapy and radiation therapy on cancer cells. Analysis of the thermobiological effects of local hyperthermia indicates that it is a potent sensitizer of cell killing by ionizing radiation and chemotherapy. The increase in tumor radiosensitivity is caused by the inhibition of the repair processes of damaged dna strands. Hyperthermia enhances perfusion and oxygenation of hypoxic tumor cells with a consecutive increase in tumor radiosensitivity. During chemotherapy, local hyperthermia ensures the maximum targeted delivery of cytotoxic agents to the tumor, thus increasing the effectiveness of treatment. Moreover, local hyperthermia has a direct cytotoxic effect on tumor cells. Randomized trials on the use of hyperthermia in the treatment of locally advanced cervical cancer have shown positive immediate and long-term treatment outcomes.Conclusion. Local hyperthermia combined with chemotherapy and radiation therapy is a promising treatment modality for locally advanced cervical cancer, because it can significantly improve treatment outcomes and reduce the frequency of early and late adverse effects. However, despite the available world experience, there are no unified methodological approaches to local hyperthermia, and therefore further research is required.

The crystal structure of human XPG, the xeroderma pigmentosum group G endonuclease, provides insight into nucleotide excision DNA repair.

Nucleotide excision repair (NER) is an essential pathway to remove bulky lesions affecting one strand of DNA. Defects in components of this repair system are at the ground of genetic diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The XP complementation group G (XPG) endonuclease cleaves the damaged DNA strand on the 3′ side of the lesion coordinated with DNA re-synthesis. Here, we determined crystal structures of the XPG nuclease domain in the absence and presence of DNA. The overall fold exhibits similarities to other flap endonucleases but XPG harbors a dynamic helical arch that is uniquely oriented and defines a gateway. DNA binding through a helix-2-turn-helix motif, assisted by one flanking α-helix on each side, shows high plasticity, which is likely relevant for DNA scanning. A positively-charged canyon defined by the hydrophobic wedge and β-pin motifs provides an additional DNA-binding surface. Mutational analysis identifies helical arch residues that play critical roles in XPG function. A model for XPG participation in NER is proposed. Our structures and biochemical data represent a valuable tool to understand the atomic ground of XP and CS, and constitute a starting point for potential therapeutic applications.

The intrinsic ATPase activity of Mycobacteriumtuberculosis UvrC is crucial for its damage-specific DNA incision function.

To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties of Mycobacteriumtuberculosis UvrABC excinuclease complex. Furthermore, the biochemical properties of Escherichiacoli UvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instability invitro. In addition, homology modelling of M.tuberculosis UvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC.

Enhanced radiotherapy using photothermal therapy based on dual-sensitizer of gold nanoparticles with acid-induced aggregation

The damaged DNA strands caused by radiotherapy (RT) can repair by themselves. A gold nanoparticles (GNPs) system with acid-induced aggregation was developed into a dual sensitizer owing to its high radioactive rays attenuation ability and enhanced photothermal heating efficiency after GNPs aggregation to achieve a combination therapy of RT and photothermal therapy (PTT). In this combination therapy, the formed GNP aggregates firstly showed a higher sensitize enhancement ratio (SER) value (1.52). Importantly, the self-repair of damaged DNA strands was inhibited by mild PTT through down-regulating the expression of DNA repair protein, thus resulting in a much higher SER value (1.68). Anti-tumor studies further demonstrated that this combination therapy exhibited ideal anti-tumor efficacy. Furthermore, the imaging signals of GNPs in computed tomography and photoacoustic were significantly improved following the GNPs aggregation. Therefore, a dual sensitizer with multimodal imaging was successfully developed and can be further applied as a new anti-tumor therapy.

Structural transitions and mechanochemical coupling in the nucleoprotein filament explain homology selectivity and Rad51 protein cooperativity in cellular DNA repair.

The nucleoprotein filament (NPF) is the fundamental element of homologous recombination (HR), a major mechanism for the repair of double-strand DNA breaks in the cell. The NPF is made of the damaged DNA strand surrounded by recombinase proteins, and its sensitivity to base-pairing mismatches is a crucial feature that guarantees the fidelity of the repair. The concurrent recombinases are also essential for several steps of HR. In this work, we used torque-sensitive magnetic tweezers to probe and apply mechanical constraints to single nucleoprotein filaments (NPFs). We demonstrated that the NPF undergoes structural transitions from a stretched to a compact state, and we measured the corresponding mechanochemical signatures. Using an active two-state model, we proposed a free-energy landscape for the NPF transition. Using this quantitative model, we explained both how the sensitivity of the NPF to the homology length is regulated by its structural transition and how the cooperativity of Rad51 favors selectivity to relatively long homologous sequences.

Deficiency in the DNA repair protein ERCC1 triggers a link between senescence and apoptosis in human fibroblasts and mouse skin.

ERCC1 (excision repair cross complementing‐group 1) is a mammalian endonuclease that incises the damaged strand of DNA during nucleotide excision repair and interstrand cross‐link repair. Ercc1−/Δ mice, carrying one null and one hypomorphic Ercc1 allele, have been widely used to study aging due to accelerated aging phenotypes in numerous organs and their shortened lifespan. Ercc1−/Δ mice display combined features of human progeroid and cancer‐prone syndromes. Although several studies report cellular senescence and apoptosis associated with the premature aging of Ercc1−/Δ mice, the link between these two processes and their physiological relevance in the phenotypes of Ercc1−/Δ mice are incompletely understood. Here, we show that ERCC1 depletion, both in cultured human fibroblasts and the skin of Ercc1−/Δ mice, initially induces cellular senescence and, importantly, increased expression of several SASP (senescence‐associated secretory phenotype) factors. Cellular senescence induced by ERCC1 deficiency was dependent on activity of the p53 tumor‐suppressor protein. In turn, TNFα secreted by senescent cells induced apoptosis, not only in neighboring ERCC1‐deficient nonsenescent cells, but also cell autonomously in the senescent cells themselves. In addition, expression of the stem cell markers p63 and Lgr6 was significantly decreased in Ercc1−/Δ mouse skin, where the apoptotic cells are localized, compared to age‐matched wild‐type skin, possibly due to the apoptosis of stem cells. These data suggest that ERCC1‐depleted cells become susceptible to apoptosis via TNFα secreted from neighboring senescent cells. We speculate that parts of the premature aging phenotypes and shortened health‐ or lifespan may be due to stem cell depletion through apoptosis promoted by senescent cells.

Knockdown of KLF5 promotes cisplatin-induced cell apoptosis via regulating DNA damage checkpoint proteins in non-small cell lung cancer.

BackgroundPrevious research has revealed that Krüppel‐like factor 5 (KLF5) may affect DNA damage repair pathways; however, the associated molecular mechanisms are unclear.MethodsThe expression of KLF5 was studied by immunohistochemical staining in paired tumour and normal tissues from 90 patients with ESCC. We studied the effects of KLF5 knockdown on cell proliferation and apoptosis with or without cisplatin treatment in A549 and H1299 cell lines. Moreover, we examined the effect of KLF5 on the DNA damage response.Results KLF5 was significantly overexpressed in non‐small cell lung cancer (NSCLC) tissues, and high KLF5 expression predicted poor prognosis for NSCLC patients. The inhibition of KLF5 markedly augmented cisplatin‐induced cell apoptosis. In addition, we observed that KLF5 knockdown could decrease DNA repair potential by inhibiting H2AX S139 phosphorylation in response to cisplatin. Moreover, silencing of KLF5 in NSCLC cell lines inhibited the phosphorylation of checkpoint kinases Chk1 S345 and Chk2 T68. KLF5 knockdown permits cells with broken or damaged DNA strands to enter mitosis by inhibiting the activation of H2AX, Chk1 and Chk2, resulting in mitotic catastrophe.Conclusion KLF5 plays a significant role in the DNA damage response by regulating DNA damage checkpoint proteins. Inhibition of KLF5 may be a potential therapeutic target for NSCLC patients with cisplatin resistance.

Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli.

BackgroundDNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2′-deoxyguanosine-5′-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage.MethodsHere, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproduct (6–4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured.ResultsWe demonstrated that MutY does not excise Adenine when it is paired with CPD and 6–4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains.DiscussionThese findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

RAD7 homologues contribute to Arabidopsis UV tolerance

Frequent exposure of plants to solar ultraviolet radiation (UV) results in damaged DNA. One mechanism of DNA repair is the light independent pathway Global Genomic Nucleotide Excision Repair (GG-NER), which repairs UV damaged DNA throughout the genome. In mammals, GG-NER DNA damage recognition is performed by the Damaged DNA Binding protein 1 and 2 (DDB1/2) complex which recruits the Xeroderma Pigmentosa group C (XPC) / RAD23D complex. In the yeast Saccharomyces cerevisiae, distinct proteins, Radiation sensitive 7 and 16 (Rad7p and Rad16p), recognize the damaged DNA strand and then recruit the XPC homologue, Rad4p, and Rad23p. The remainder of the proteins involved GG-NER are well conserved. DDB1, DDB2, XPC/RAD4, and RAD23 homologues have been described in the model plant Arabidopsis thaliana. In this study we characterize three Arabidopsis RAD7 homologues, RAD7a, RAD7b, and RAD7c. Loss of function alleles of each of the three RAD7 homologues result in increased UV sensitivity. In addition, RAD7b and RAD7c overexpression lines exhibited increased UV tolerance. Thus RAD7 homologues contribute to UV tolerance in plants as well as in yeast. This is the first time any system has been shown to utilize both the DDB1/2 and RAD7/16 damage recognition complexes.

Activation of the DNA-repair mechanism through NBS1 and MRE11 diffusion.

The non-homologous end joining of a DNA double strand break is initiated by the MRE11-NBS1-RAD50 complex whose subunits are the first three proteins to arrive to the breakage site thereby making the recruitment time of MRE11, NBS1 and RAD50 essential for cell survival. In the present investigation, the nature of MRE11 and NBS1 transportation from the cytoplasm to the nucleus, hosting the damaged DNA strand, is hypothesized to be a passive diffusive process. The feasibility of such a mechanism is addressed through theoretical and computational approaches which permit establishing the characteristic recruitment time of MRE11 and NBS1 by the nucleus. A computational model of a cell is constructed from a set of biological parameters and the kinetic Monte Carlo algorithm is used to simulate the diffusing MRE11 and NBS1 particles as a random walk process. To accurately describe the experimented data, it is discovered that MRE11 and NBS1 should start diffusion from significantly different starting positions which suggests that diffusion might not be the only transport mechanism of repair protein recruitment to the DNA break.

RNA polymerase II is released from the DNA template during transcription-coupled repair in mammalian cells

In mammalian cells, bulky DNA adducts located in the template but not the coding strand of genes block elongation by RNA polymerase II (RNAPII). The blocked RNAPII targets these transcription-blocking adducts to undergo more rapid excision repair than adducts located elsewhere in the genome. In excision repair, coupled incisions are made in the damaged DNA strand on both sides of the adduct. The fate of RNAPII in the course of this transcription-coupled repair (TCR) pathway is unclear. To address the fate of RNAPII, we used methods that control transcription to initiate a discrete "wave" of elongation complexes. Analyzing genome-wide transcription and repair by next-generation sequencing, we identified locations of elongation complexes and transcription-repair coupling events in genes throughout the genome. Using UV-exposed human skin fibroblasts, we found that, at the dose used, a single wave of elongation complexes was blocked within the first 25 kb of genes. TCR occurred where the elongation complexes were blocked, and repair was associated with the dissociation of these complexes. These results indicate that individual elongation complexes do not engage in multiple rounds of TCR with successive lesions. Our results are consistent with a model in which RNAPII is dissociated after the dual incision of the transcription-blocking lesion, perhaps by Cockayne syndrome group B translocase, or during the synthesis of a repair patch.

Polymorphisms in XPB and XPG subcomplexes of TFIIH is associated with lung cancer risk in a Chinese population: A case-control study

Objective: The transcription factor IIH (TFIIH) helicases XPB and XPD are responsible for opening the DNA strand around the lesion site and endonuclease XPG cleaves the damaged DNA strand on the 3’ side during nucleotide excision repair (NER). Polymorphisms in these three genes that affect DNA repair capacity may contribute to susceptibility of lung cancer. In this study, our objective is to conduct a case-control study of 100 Chinese patients with lung cancer and 100 cancer-free age and sex matched controls to analyse associations between these SNPs and lung cancer susceptibility. Methods: In this hospital-based case-control study, we genotyped 7 SNPs of XPB, XPD and XPG using matrix assisted laser desorption ionisation-time of flight mass spectrometry method (MALDI-TOF) to explore the association with lung cancer risk. To estimate the relative risk of lung cancer associated with SNP genotype, odds ratios (OR) and 95.0% confidence intervals (95.0% CI) were obtained from unconditional multinomial logistic regression models without and with adjustment for potential confounders including age, gender, cigarette smoking and alcohol consumption. Results: The results showed that individuals carrying XPB rs4150434 GA or AA genotype (OR per GA genotype, 1.997; 95.0% CI: 1.031-3.871; P=0.039; OR per AA genotype, 2.435; 95.0% CI: 1.037-5.718; P=0.037), and this association was also find in nondrinkers (OR per GA genotype, 2.477; 95.0% CI: 1.128-5.440; P=0.022). Individuals carrying XPG rs2094258 AA genotype had an increased risk of lung cancer (OR per AA genotype, 3.020; 95.0% CI: 1.015-8.980; P=0.040) compared with individuals with the GG genotype, especially in nondrinkers (OR per AA genotype, 4.020; 95.0% CI: 1.211-13.339; P=0.017). In addition, we found that XPG rs17655 CG or GG genotype associated with decreased lung cancer risk in drinkers (OR per XPG rs17655 CG genotype, 0.238; 95.0% CI: 0.061~0.925; P=0.034; OR per XPG rs17655 GG genotype, 0.l39; 95.0% CI: 0.021-0.938; P=0.032). Haplotype analysis of all 7 SNPs was also conducted. We found that the haplotype of XPB (rs4150441, G>A; rs4150434, G>A) GA and the haplotype of XPG (rs2094258, G>A; rs4771436, T>G; rs17655, C>G) ATC had an increased association with lung cancer. Conclusions: These findings suggest an important role of XPB rs4150434 and XPG rs17655 polymorphisms for a biomarker for lung cancer risk among the Chinese population.

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit.

Isolated human ribosomal protein uS3 has extra-ribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3΄ to the abasic (AP) site. However, the ability of uS3 residing within ribosome to recognize and cleave damaged DNA has never been addressed. Here, we compare interactions of single-stranded (ss) DNA and dsDNA bearing AP site with human ribosome-bound uS3 and with the isolated protein, whose interactions with ssDNA were not yet studied. The AP lyase activity of free uS3 was much higher with ssDNA than with dsDNA, whereas ribosome-bound uS3 was completely deprived of this activity. Nevertheless, an exposed peptide of ribosome-bound uS3 located far away from the putative catalytic center previously suggested for isolated uS3 cross-linked to full-length uncleaved ssDNA, but not to dsDNA. In contrast, free uS3 cross-linked mainly to the 5΄-part of the damaged DNA strand after its cleavage at the AP site. ChIP-seq analysis showed preferential uS3 binding to nucleolus-associated chromatin domains. We conclude that free and ribosome-bound uS3 proteins interact with AP sites differently, exhibiting their non-translational functions in DNA repair in and around the nucleolus and in regulation of DNA damage response in looped DNA structures, respectively.

PostExcision Events in Human Nucleotide Excision Repair

The nucleotide excision repair system removes a wide variety of DNA lesions from the human genome, including photoproducts induced by ultraviolet (UV) wavelengths of sunlight. A defining feature of nucleotide excision repair is its dual incision mechanism, in which two nucleolytic incision events on the damaged strand of DNA at sites bracketing the lesion generate a damage-containing DNA oligonucleotide and a single-stranded DNA gap approximately 30 nucleotides in length. Although the early events of nucleotide excision repair, which include lesion recognition and the dual incisions, have been explored in detail and are reasonably well understood, the fate of the single-stranded DNA gaps and excised oligonucleotide products of repair have not been as extensively examined. In this review, recent findings that address these less-explored aspects of nucleotide excision repair are discussed and support the concept that postincision gap and excised oligonucleotide processing are critical steps in the cellular response to DNA damage induced by UV light and other environmental carcinogens. Defects in these latter stages of repair lead to cell death and other DNA damage signaling responses and may therefore contribute to a number of human disease states associated with exposure to UV wavelengths of sunlight, including skin cancer, aging and autoimmunity.