Nucleotide Excision Repair Enzymes Research Articles

Reduction of selenite to selenium nanoparticles by highly selenite-tolerant bacteria isolated from seleniferous soil

The microbial reduction of selenite to elemental selenium nanoparticles (SeNPs) is thought to be an effective detoxification process of selenite for many bacteria. In this study, Metasolibacillus sp. ES129 and Oceanobacillus sp. ES111 with high selenite reduction efficiency or tolerance were selected for systematic and comparative studies on their performance in selenite removal and valuable SeNPs recovery. The kinetic monitoring of selenite reduction showed that the highest transformation efficiency of selenite to SeNPs was achieved at a concentration of 4.24 mM for ES129 and 4.88 mM for ES111. Ultramicroscopic analysis suggested that the SeNPs produced by ES111 and ES129 had been formed in cytoplasm and subsequently released to extracellular space through cell lysis process. Furthermore, the transcriptome analysis indicated that the expression of genes involved in bacillithiol biosynthesis, selenocompound metabolism and proline metabolism were significantly up-regulated during selenite reduction, suggesting that the transformation of selenite to Se0 may involve multiple pathways. Besides, the up-regulation of genes associated with nucleotide excision repair and antioxidation-related enzymes may enhance the tolerance of bacteria to selenite. Generally, the exploration of selenite reduction and tolerance mechanisms of the highly selenite-tolerant bacteria is of great significance for the effective utilization of microorganisms for environmental remediation.

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

Chemical mutagenesis of Listeria monocytogenes for increased tolerance to benzalkonium chloride shows independent genetic underpinnings and off-target antibiotic resistance.

Listeria monocytogenes, a potentially fatal foodborne pathogen commonly found in food processing facilities, creates a significant economic burden that totals more than $2 billion annually in the United States due to outbreaks. Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC), are among the most widely used sanitizers to inhibit the growth and spread of L. monocytogenes from food processing facilities. However, resistance to QACs has been increasing in L. monocytogenes and different genetic mechanisms conferring resistance have been discovered. Here, we used ethyl methanesulfonate (EMS) to chemically mutagenize the BAC-susceptible strain, L. monocytogenes FSL-N1-304. We isolated two mutants with increased tolerance to BAC compared to the parental strain. Next, we assessed the off-target effect of increased tolerance to BAC by measuring the minimum inhibitory concentrations (MICs) of a diverse set of antibiotics, revealing that mut-1 and mut-2 displayed significantly increased resistance to fluoroquinolone antibiotics compared to the parental strain. A hemolysis assay was then used to investigate a potential correlation between BAC tolerance and virulence. Interestingly, mut-1 and mut-2 both exhibited significantly higher hemolysis percentage than the parental strain. We then sequenced the genomes of the parental strain and both mutants to identify mutations that may be involved in the increased resistance to BAC. We identified 3 and 29 mutations in mut-1 and mut-2, respectively. mut-1 contained nonsynonymous mutations in dagK (a diacylglycerol kinase), lmo2768 (a permease-encoding gene), and lmo0186 (resuscitation promoting factor). mut-2 contained a nonsense mutation in the nucleotide excision repair enzyme UvrABC system protein B encoding gene, uvrB, which likely accounts for the higher number of mutations observed. Transcriptome analysis in the presence of BAC revealed that genes related to the phosphotransferase system and internalins were up-regulated in both mutants, suggesting their significance in the BAC stress response. These two mutants provide insights into alternative mechanisms for increased BAC tolerance and could further our understanding of how L. monocytogenes persists in the food processing environment.

Crucial role and mechanism of transcription-coupled DNA repair in bacteria.

Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype1-3. Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo. We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.

DNA Repair Capacity for Personalizing Risk and Treatment Response − Assay Development and Optimization in Human Peripheral Blood Mononuclear Cells (PBMCs)

DNA repair capacity (DRC) is the ability of a cell to repair DNA damage. Differential DRC plays an important role in human disease, including lung and other cancers. Measuring DRC could aid in translational disease research and in personalizing treatment. We developed and optimized a flow cytometry-based assay to measure individual DRC using GFP-expressing plasmids modified by ultraviolet (UV) light for nucleotide excision repair (NER) and restriction enzyme digestion to induce a blunt double-strand cut between promoter and GFP expression regions for nonhomologous end joining (NHEJ). Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy volunteers were used to measure DRC and optimize the assay. Pathway specificity of the NHEJ DRC assay was confirmed using Ku80-/- MEF cells, which showed a 6-fold reduction in NHEJ compared to Ku80+/+. Using a cell mixing assay, we show a linear correlation between NHEJ DRC and the expected concentration of Ku80. NHEJ DRC measurements in cryopreserved PBMCs are quantifiable with low interindividual and inter-assay variability, and a titratable decrease in NHEJ activity was observed in PBMCs treated with the DNA-PK inhibitor NU7441. Pathway specificity of the NER DRC assay was confirmed by a decrease in measured NER activity in human XPC deficient compared to XPC proficient fibroblasts, with a linear correlation measured between NER DRC and expected XPC concentration by cell mixing assay. NER DRC is quantifiable, reproducible, and titratable in PBMCs from healthy volunteers. We measured both NER and NHEJ DRC in PBMCs obtained from newly diagnosed, untreated lung cancer patients; measured DRC differed in these PBMCs compared to healthy volunteers. With further investigation, measurement of NER and NHEJ DNA repair capacity may be useful in personalizing disease risk and response to DNA damaging therapies and small molecular inhibitors of DNA repair pathways using readily available human PBMCs.

ERCC5 quantification for age estimation of peripheral blood in a Chinese Han population.

Age estimation is an important topic of human identification in forensic practice, especially coming to biological samples in crime scene, such as blood, saliva, semen. As rate-limiting enzyme in Nucleotide excision repair (NER) that was associated with aging, Excision repair cross-complementation group 5 (ERCC5) was considered to be a candidate biomarker for individual age estimation. The ERCC5 mRNA and protein expression levels association with age have been demonstrated in our previous study. However, very little is known about relationship DNA-based quantification of ERCC5 with age. In this study, we detected ERCC5 level in peripheral blood from a Chinese Han population by SYBR qPCR assay to gain better insight into the quantitative relationship with age. The results showed ERCC5 level declined with individual age with a negative correlation(r=-0.8, R2=0.63, P<0.001). The data model for age estimation based on ERCC5 level was Y=-31.352X+14.436±10.28 (Y: age, year; X: CqTBP-CqERCC5; standard error: year). The accuracy about the data model for age estimation was about 73.33%. The mean absolute difference (MAD) values were 8.22, 8.09 and 8.38 in total, male and female, respectively. Furthermore, ERCC5 quantification for age estimation was also applicable for stored blood samples under low temperature up to 6months. It was suggested that the ERCC5 quantification was expected to be a valuable additional method for individual age estimation, especially in cases where traditional morphologic method is absent or inefficient in forensic practice.

Helicobacter pylori severely reduces expression of DNA repair proteins PMS2 and ERCC1 in gastritis and gastric cancer

Gastric cancers are the third leading cause of cancer mortality in the world. Helicobacter pylori causes over 60 % of all stomach cancers. Colonization of the gastric mucosa by H. pylori results in increased DNA damage. Repair of DNA damage may also be reduced by H. pylori infection. Reduced DNA repair in combination with increased DNA damage can cause carcinogenic mutations. During progression to gastric cancer, gastric epithelium goes through stages of increasing pathology. Determining the levels of DNA repair enzymes during progression to gastric cancer could illuminate treatment approaches. Our aim is to determine the level of gastric expression of DNA repair proteins ERCC1 (a nucleotide excision repair enzyme) and PMS2 (a mismatch repair enzyme) in the presence of H. pylori infection at successive stages of gastric pathology and in gastric cancers. We analyzed gastric tissues of 300 individuals, including 30 without dyspepsia, 200 with dyspepsia and 70 with gastric cancers. The presence of H. pylori, gastric pathology and expression of DNA repair proteins ERCC1 and PMS2 were evaluated.Infection by H. pylori carrying the common cagA gene reduced median nuclear expression of ERCC1 and PMS2 to less than 20 % and 15 % of normal, respectively, in all pathologic stages preceding cancer.ERCC1 and PMS2 nuclear expression was 0–5 % of normal in gastric cancers. H. pylori can cause deficiency of ERCC1 and PMS2 protein expression. These deficiencies are associated with gastric pathology and cancer. This reduction in DNA repair likely causes carcinogenic mutations. Substantially reduced ERCC1 and PMS2 expression appears to be an early step in progression to H. pylori-induced gastric cancer.

561 Time-dependence of UVB induced cellular mechanisms in cultured human keratinocytes

UVB-induced cyclobutane pyrimidine dimers (CPDs) are considered to be the main cause of acute sunburn and epidermal carcinogenesis. In humans, these lesions are repaired by nucleotide excision repair, but marsupials and lower organisms present photolyase enzyme which rapidly removes CPDs in a visible light-dependent process (photoreactivation). Previously we established an in vitro pseudouridin-modified mRNA encoding CPD-specific photolyase transfection on human keratinocyte cell lines, which was proved to be a proper method to avoid UVB-induced apoptosis. According to clinical experiences, there is a need for a treatment to diminish the deleterious effects of sunburn when UVB injury has already occurred and the first symptoms appear. Our aim was to determine the time interval after UVB exposure when keratinocyte apoptosis is still preventable. Normal human epidermal keratinocytes were transfected with lipofectamine-complexed, in vitro transcribed mRNA encoding CPD-specific photolyase. Cells were irradiated with 60 mJ/cm2 UVB. At 0, 6, 8 or 12 hours after UVB cells were either exposed to visible light (photoreactivation) or kept in the dark. Viability was measured by Annexin V and propidium iodide dual staining followed by flow cytometry, the relative amount of intracellular CPDs was analyzed by CPD-specific ELISA. Photolyase-mediated CPD removal restored cell viability close to the baseline conditions 0 to 6 hours after UVB treatment. The effect of CPD removal on keratinocyte survival began to decrease 8 hours after the UVB damage. In mRNA-transfected and photoreactivated cells 80% of CPDs were removed at 6 hours post-irradiation. 8 to 12 hours after UVB 40-60% of the CPDs were eliminated. Our results suggest that UV-induced keratinocyte apoptosis can be prevented within 6-8 hours after the UVB injury by the elimination of CPD photolesions. After that point the effect of CPD removal on cell viability is less feasible.

Mechanism of DNA Lesion Homing and Recognition by the Uvr Nucleotide Excision Repair System.

Nucleotide excision repair (NER) is an essential DNA repair system distinguished from other such systems by its extraordinary versatility. NER removes a wide variety of structurally dissimilar lesions having only their bulkiness in common. NER can also repair several less bulky nucleobase lesions, such as 8-oxoguanine. Thus, how a single DNA repair system distinguishes such a diverse array of structurally divergent lesions from undamaged DNA has been one of the great unsolved mysteries in the field of genome maintenance. Here we employ a synthetic crystallography approach to obtain crystal structures of the pivotal NER enzyme UvrB in complex with duplex DNA, trapped at the stage of lesion-recognition. These structures coupled with biochemical studies suggest that UvrB integrates the ATPase-dependent helicase/translocase and lesion-recognition activities. Our work also conclusively establishes the identity of the lesion-containing strand and provides a compelling insight to how UvrB recognizes a diverse array of DNA lesions.

Excision repair cross-complementing group 1 (ERCC1) overexpression inhibits cell apoptosis and is associated with unfavorable prognosis of esophageal squamous cell carcinoma.

Excision repair cross-complementing group 1 (ERCC1) functions as a nucleotide excision repair (NER) enzyme. Altered ERCC1 expression or function is closely associated with cancer development and progression. This study determined the association of ERCC1 expression with survivin expression, clinicopathological characteristics, and survival of esophageal squamous cell carcinoma (ESCC) patients after postoperative concurrent chemoradiotherapy.Tissue specimens from 102 resected ESCC patients were acquired for immunohistochemical analysis of ERCC1 and survivin protein expression.ERCC1 expression was detected in 62.7% of ESCC tissues and in 9.8% of normal squamous epithelium tissues (P < .01), while survivin expression was detected in 60.8% of ESCC tissues and in 19.6% of normal squamous epithelia (P < .01). ERCC1 overexpression associated with advanced tumor clinical stage and lymph node metastasis (P < .05), but not with tumor size, depth of invasion, or differentiation (P > .05). ERCC1 overexpression was also associated with survivin levels (r = 0.42, P < .01) and worse progression-free survival of ESCC patients after concurrent chemoradiotherapy. Multivariate analysis data revealed that ERCC1 and survivin protein expression were independent predictors of overall survival of ESCC patients after chemotherapy and/or radiotherapy (P < .05).ERCC1 overexpression is an important phenotype that is associated with ESCC lymph node metastasis and advanced tumor clinical stages. ERCC1 expression may also inhibit ESCC cell apoptosis via regulating survivin expression, and ERCC1 and survivin overexpression are independent predictors of prognosis for ESCC patients who receive chemotherapy and/or radiotherapy.

The age-related expression decline of ERCC1 and XPF for forensic age estimation: A preliminary study

The age-related capacity decline of DNA damage repair in human peripheral blood has been demonstrated. Excision repair cross-complementation group1 (ERCC1) and Xeroderma pigmentosum complementation group F (XPF) were rate-limiting enzyme in nucleotide excision repair (NER) which was known as the most important DNA damage repair system. Consequently, we hypothesized that the expression and/or activity of ERCC1 and XPF may be associated with age. However, little was known about the quantitative relationship of ERCC1 and XPF expression levels with age. The aim of the present study was to analyze the correlation of ERCC1 and XPF expression levels with age by detecting the ERCC1 and XPF mRNA levels in peripheral blood mononuclear cells(PBMCs) and protein levels in plasma in healthy ethnic Han Chinese individuals, and finally find new molecular markers for forensic age estimation by establishing the mathematical model between ERCC1 and XPF expression levels and age. The results showed that the ERCC1 and XPF mRNA relative expression levels in PBMCs declined in an age-dependent manner (r = −0.578/−0.844, respectively, P < 0.01). The formula for age estimation based on the ERCC1 and XPF mRNA relative expression levels decline in PBMCs were Y = 3.3E−5x2−0.0261x+1.9175 (R2 = 0.3244, P < 0.01) and Y = 0.0003x2−0.0459x+2.0439 (R2 = 0.729, P < 0.01), respectively. There were no significant differences of the ERCC1 or XPF protein expression levels in plasma between age groups (P > 0.05). Furthermore, there were no significant differences of the ERCC1 or XPF mRNA and/or protein expression levels between males and females(P > 0.05). It suggested that the ERCC1 and XPF mRNA expression levels could be considered as valuable additional tool in individual age estimation, especially in cases where traditional morphologic method was inefficient or absent in forensic practice.

Molecular mechanisms of discrotophos-induced toxicity in HepG2 cells: The role of CSA in oxidative stress

Dicrotophos (Dic), an insecticide and acaricide, is used against a variety of sucking, boring and chewing pests. It was proven that Dic induced oxidative DNA damage in HepG2 cells. However, the molecular mechanisms of this compound were still unclear. First of all, the cytotoxicity and oxidative DNA damage were confirmed. Next, using RNA-seq for detecting differential expressed genes (DEGs) in cells treated with 50 μM Dic for 24 h, we showed that the dysregulation of these genes, irrespective of up (1298 genes) or down (2125 genes) regulation, could be attributed to some diverse pathways/metabolisms using KEGG analysis, particularly in DNA damage responses (DDRs) such as oxidative phosphorylation, nucleotide excision repair and cell cycle arrest. Validation of some randomly selected DDR genes confirmed RNA-seq results. We further demonstrated that Dic induced ROS overproduction, the loss of mitochondrial depolarization and cell cycle arrest in the G0/G1 phase. In addition, we also definitely clarified the role of CSA, a nucleotide excision repair enzymes in Dic-treated cells. Collectively, our results showed that various mechanisms of Dic-induced toxicity in HepG2 cells including downregulation of some genes related to nucleotide excision repair including CSA and increased oxidative stress.

Emerging roles for histone modifications in DNA excision repair

DNA repair is critical to maintain genome stability. In eukaryotic cells, DNA repair is complicated by the packaging of the DNA 'substrate' into chromatin. DNA repair pathways utilize different mechanisms to overcome the barrier presented by chromatin to efficiently locate and remove DNA lesions in the genome. DNA excision repair pathways are responsible for repairing a majority of DNA lesions arising in the genome. Excision repair pathways include nucleotide excision repair (NER) and base excision repair (BER), which repair bulky and non-bulky DNA lesions, respectively. Numerous studies have suggested that chromatin inhibits both NER and BER in vitro and in vivo Growing evidence demonstrates that histone modifications have important roles in regulating the activity of NER and BER enzymes in chromatin. Here, we will discuss the roles of different histone modifications and the corresponding modifying enzymes in DNA excision repair, highlighting the role of yeast as a model organism for many of these studies.

Purine 5′,8-cyclo-2′-deoxynucleoside lesions in irradiated DNA

Having their position gained among the smallest bulky DNA lesions recognized by the nucleotide excision repair (NER) enzyme, purine 5′,8-cyclo-2′-deoxynucleosides (5′,8-cPu) are increasingly attracting the interest in the field of genome integrity in health and diseases. Exclusively generated by one of the most harmful of the reactive oxygen species, the hydroxyl radical, 5′,8-cPu can be utilized also for highly valuable information regarding the oxidative status nearby the area where the genetic information is stored. Herein, we have collected the most recently reported biological studies, focusing on the repair mechanism of these lesions and their biological significance particularly in transcription. The LC-MS/MS quantification protocols that appeared in the literature are discussed in details, along with the reported values for the four 5′,8-cPu produced by in vitro γ-radiolysis experiments with calf thymus DNA. Mechanistic insights in the formation of the purine 5′,8-cyclo-2′-deoxynucleosides and their chemical stability are also given in the light of their potential to be utilized as DNA biomarkers of oxidative stress.

XPG Promotes Homologous Recombination Repair and Genomic Stability

Abstract The nucleotide excision repair enzyme XPG has a noncatalytic role in HRR and genomic stability.

A molecular biomarker targeted approach to adjuvant therapy for resected pancreatic adenocarcinoma: Results of a phase II prospective trial.

230 Background: Standard adjuvant treatment for resected pancreatic adenocarcinoma is gemcitabine (Gem) (CONKO-001 trial: Gem vs placebo DFS 13.4 vs 6.7 mo; p&lt;0.001; OS 22.8 vs 20.2 mo; p=0.01). Adding cisplatin (Cis) to Gem has shown increased response rates in the metastatic setting. This benefit may be inhibited by high expression of excision repair cross-complementing gene–1 (ERCC1), the key enzyme in nucleotide excision repair. This Phase II prospective trial assesses outcomes of patients treated with adjuvant Gem/Cis chemotherapy, stratifying results by tumor ERCC1 expression. Methods: Patients with resected pancreatic adenocarcinoma at a single institution were enrolled from 2010-2013. Initially, patients received Gem (1000 mg/m2) / Cis (50 mg/m2) Day 1/8/15, Q28d for 6 cycles. After enrolling 5 pts, this was modified to Day 1/15 due to toxicity. Two dose reductions were permitted. Intent to treat analyses were conducted. Tumor ERCC1 expression was evaluated by immunohistochemistry and dichotomized into low and high expression groups. Primary outcomes were RFS and OS stratified by ERCC1 expression. Results: Of 22 pts, 16 (73%) had Stage IIB disease, 5 (23%) Stage IIA, and 1 (4%) Stage IA. Thirteen (59%) completed all 6 cycles of therapy, of whom 9 required dose reduction. Of the remaining 9 pts, 4 completed &gt;68% of intended therapy. Grade 3 and 4 toxicity occurred in 13 pts (59%); neutropenia was most common (n=9;41%). Median follow-up was 37.5 mo. Median RFS was 16.7 mo, and OS was 35.5 mo. ERCC1 tumor expression data were available for 20 pts: 15 low (75%) and 5 high (25%). Low compared to high ERCC1 was not associated with improved RFS (12.4 vs 16.7 mo; p=0.68) or OS (Median not reached vs 21.6 mo; p=0.22). Conclusions: Adjuvant gemcitabine/cisplatin is tolerated by patients with resected pancreatic adenocarcinoma. RFS and OS for Gem/Cis appear promising compared to historic control. Tumor ERCC1 expression can be reliably evaluated, and low expression is present in the majority of patients. Further prospective trials evaluating Gem/Cis as an adjuvant regimen and ERCC-1 as a biomarker in resected pancreatic adenocarcinoma are warranted. Clinical trial information: NCT01188109.

Protein expression of DNA damage repair proteins dictates response to topoisomerase and PARP inhibitors in triple-negative breast cancer.

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.

Xeroderma pigmentosum: Composite cutaneous malignancies A nightmare

Xeroderma pigmentosum (XP), is an autosomal recessive genetic disorder of enzymes in which the ability to repair damage caused to DNA by UV light is deficient. As this disorder is commonly due to exposure to sunlight and requires protection from sunlight such patients with the disease are often commonly referred to as “children of the night”. In this disorder, the patients suffer from various cutaneous malignancies such as basal cell carcinomas (BCC), malignant melanoma (MM), and squamous cell carcinoma (SCC). It is a rare disorder with an incidence of 1:250,000 in the United States. This disease involves both sexes and all races, with a common occurrence in Japanese people. The disease is characterized by a mutation in nucleotide excision repair (NER) enzymes resulting in a reduction or elimination of NER which is required for repair of UV light induced deoxyribonucleic acid damage thus increasing susceptibility to cutaneous malignancies. These patients rarely survive beyond 20 years of age. This case reports a rare occurrence of three malignancies in a single tumor BCC, SCC, and the rarely occurring sebaceous carcinoma, which is not known to be associated with UV radiation exposure.

NER enzymes maintain genome integrity and suppress homologous recombination in the absence of exogenously induced DNA damage in Pseudomonas putida.

In addition to its prominence in producing genetic diversity in bacterial species, homologous recombination (HR) plays a key role in DNA repair and damage tolerance. The frequency of HR depends on several factors, including the efficiency of DNA repair systems as HR is involved in recovery of replication forks perturbed by DNA damage. Nucleotide excision repair (NER) is one of the major DNA repair pathways involved in repair of a broad range of DNA lesions generally induced by exogenous chemicals or UV-irradiation and its functions in the cells not exposed to DNA-damaging agents have attracted less attention. In this study we have developed an assay that enables to investigate HR between chromosomal loci of the soil bacterium Pseudomonas putida both in growing and stationary-phase cells. The present assay detects HR events between two non-functional alleles of phenol degrading genes that produce a functional allele and allow the growth of bacteria on phenol as a sole carbon source. Our results indicate that HR between chromosomal loci takes place mainly in the growing cells and the frequency of HR is reduced during the following starvation in NER-proficient P. putida but not in the case when bacteria lack UvrA or UvrB enzymes. The absence of UvrA or UvrB resulted in a hyper-recombination phenotype in P. putida, the cells were filamented and their growth was impaired even in the absence of exogenous DNA damage. However, NER-deficient derivatives that overcame growth defects emerged rapidly. Such adaptation resulted in the decline of the frequency of HR. Although HR in actively replicating P. putida was still elevated in the adapted variants of the UvrA- and UvrB-deficient strains, the dynamics of emergence of the recombinants in these strains turned similar to NER-proficient bacteria. Additionally, we observed that HR was enhanced in the absence of the transcription repair coupling factor Mfd in growing cells but not during starvation. The frequency of HR was not affected by the UvrA homologue UvrA2 neither in NER-proficient bacteria nor in the absence of UvrA, suggesting a minor role of UvrA2 in NER. Thus, we conclude that NER functions are important also without exogenously induced DNA damage in P. putida and both transcription-coupled and global genome NER act to suppress HR in growing cells, whereas UvrA and UvrB are involved in the maintenance of the genome integrity also in stationary-phase cells.

Reprogramming the Mechanism of Action of Chlorambucil by Coupling to a G-Quadruplex Ligand

The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low μM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.