Broad Spectrum Of DNA Lesions Research Articles

The small CRL4CSA ubiquitin ligase component DDA1 regulates transcription-coupled repair dynamics

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.

XPC–PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein.

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

XPA: DNA Repair Protein of Significant Clinical Importance.

The nucleotide excision repair (NER) pathway is activated in response to a broad spectrum of DNA lesions, including bulky lesions induced by platinum-based chemotherapeutic agents. Expression levels of NER factors and resistance to chemotherapy has been examined with some suggestion that NER plays a role in tumour resistance; however, there is a great degree of variability in these studies. Nevertheless, recent clinical studies have suggested Xeroderma Pigmentosum group A (XPA) protein, a key regulator of the NER pathway that is essential for the repair of DNA damage induced by platinum-based chemotherapeutics, as a potential prognostic and predictive biomarker for response to treatment. XPA functions in damage verification step in NER, as well as a molecular scaffold to assemble other NER core factors around the DNA damage site, mediated by protein–protein interactions. In this review, we focus on the interacting partners and mechanisms of regulation of the XPA protein. We summarize clinical oncology data related to this DNA repair factor, particularly its relationship with treatment outcome, and examine the potential of XPA as a target for small molecule inhibitors.

Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD)

Environmental exposures, reactive by-products of cellular metabolism, and spontaneous deamination events result in a spectrum of DNA adducts that if un-repaired threaten genomic integrity by inducing mutations, increasing instability, and contributing to the initiation and progression of cancer. Assessment of DNA adducts in cells and tissues is critical for genotoxic and carcinogenic evaluation of chemical exposure and may provide insight into the etiology of cancer. Numerous methods to characterize the formation of DNA adducts and their retention for risk assessment have been developed. However, there are still significant drawbacks to the implementation and wide-spread use of these methods, because they often require a substantial amount of biological sample, highly specialized expertise and equipment, and depending on technique, may be limited to the detection and quantification of only a handful of DNA adducts at a time. There is a pressing need for high throughput, easy to implement assays that can assess a broad spectrum of DNA lesions, allowing for faster evaluation of chemical exposures and assessment of the retention of adducts in biological samples. Here, we describe a new methodology, Repair Assisted Damage Detection (RADD), which utilizes a DNA damage processing repair enzyme cocktail to detect and modify sites of DNA damage for a subsequent gap filling reaction that labels the DNA damage sites. This ability to detect and label a broad spectrum of DNA lesions within cells, offers a novel and easy to use tool for assessing levels of DNA damage in cells that have been exposed to environmental agents or have natural variations in DNA repair capacity.

Cancer genomics: new software tools making sequencing more accessible.

Next-generation sequencing transforms today’s cancer research Next-generation sequencing (NGS) technologies can parallelize sequencing processes and produce millions of short-read sequences concurrently. The advent of NGS technologies has transformed today’s cancer genome research by providing an unbiased and comprehensive method of detecting somatic genome alterations [1]. The application of NGS technologies, through whole-genome, -exome and -transcriptome approaches has led to the discovery of mutated genes that drive oncogenic phenotypes in tumors. In a recent The Cancer Genome Atlas (TCGA) Pan-Cancer effort [2], 127 mutated driver genes were identified from 3281 tumors across 12 tumor types. These cancer drivers converged on several well-known pathways such as cell cycle and MAPK pathways. Interestingly, these drivers are also involved in cellular processes in cancers that have previously been less well characterized such as histone modification, RNA splicing, cellular metabolism and proteolysis. Mutations in transcription factors are more tissue specific, whereas mutations in histone modifiers are shared among multiple cancer types. The average number of driver mutations varies across tumor types and most individual tumors have two to six mutations [2]. Generally, larger numbers of driver mutations are involved in tumors with high levels of background mutations [3]. For example, most squamous cell lung carcinomas are attributed to life-long tobacco exposure. Carcinogens from tobacco exposure can cause a broad spectrum of DNA lesions on the genome, and this increases the chance that mutations conferring a small growth advantage to cells are selected in the lung microenvironment. Many such mutations with small effects can accumulate in a specific group of cells over time and collectively lead to carcinogenesis. This is in contrast to hematologic cancers, where fewer driver mutations (with potentially large effects) are observed in tumors with low background mutations. Driver genes are promising drug targets and their discovery has the potential to revolutionize personalized gene-targeted treatments that guide patient therapy according to the genomic profile of the tumor. The battle of Dr Lukas Wartman at Washington University in St Louis (WUSTL; MO, USA) against leukemia is a prime example of the promise of personalized medicine [4]. When Wartman had relapsed a second time, his colleagues at WUSTL sequenced the entire genome of DNA and RNA from his cancerous cells. His RNA sequencing showed that a gene called FLT3 was unexpectedly overproduced by his cancer cells. It so happens that the drug Sutent, previously approved for treating advanced kidney cancer, effectively inhibits FLT3. Sutent was prescribed to Wartman, and his leukemia was in remission 2 weeks later. In next decade, we will witness many such cases come to the forefront of cancer treatment and soon realize the promise of personalized medicines purred by advances in cancer genomics and associated drug development. part of Editorial

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

Poly (ADP-ribose) polymerase-1 (PARP-1) has an important role in the cellular response to a broad spectrum of DNA lesions. PARP-1 is strongly activated in response to double-stranded DNA breaks (DSBs), yet its contribution to the DSB response is poorly understood. Here we used bleomycin, a radiomimetic that generates DSBs with high specificity to focus on the response of PARP-1 to DSBs. We report that the induction of PARP-1 activity by bleomycin depends on the Ku antigen, a nonhomologous-DNA-End-Joining factor and protein phosphatase 5 (PP5). PARP-1 activation in response to bleomycin was reduced over 10-fold in Ku-deficient cells, whereas its activation in response to U.V. was unaffected. PARP-1 activation was rescued by reexpression of Ku, but was refractory to manipulation of DNA-dependent protein kinase or ATM. Similarly, PARP-1 activation subsequent to bleomycin was reduced 2-fold on ablation of PP5 and was increased 5-fold when PP5 was overexpressed. PP5 seemed to act directly on PARP-1, as its basal phosphorylation was reduced on overexpression of PP5, and PP5 dephosphorylated PARP-1 in vitro. These results highlight the functional importance of Ku antigen and PP5 for PARP-1 activity subsequent to DSBs.

Structural and Mutational Analyses of Deinococcus radiodurans UvrA2 Provide Insight into DNA Binding and Damage Recognition by UvrAs

UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms. These structures, coupled with mutational analyses and comparison with the crystal structure of class I UvrA from Bacillus stearothermophilus, suggest a previously unsuspected role for the identified insertion domains of UvrAs in both DNA binding and damage recognition. Taken together, the available information suggests a model for how UvrA interacts with DNA and thus sheds new light on the molecular mechanisms underlying the role of UvrA in the early steps of NER.

Expression of genes involved in nucleotide excision repair and sensitivity to cisplatin and melphalan in human cancer cell lines.

DNA repair has been proposed to be an important determinant of cancer cell sensitivity to alkylating agents and cisplatin (DDP). Nucleotide excision repair (NER), which represents one of the most important cellular DNA repair processes able to remove a broad spectrum of DNA lesions, is involved in the recognition and repair of the crosslinks caused by DDP and melphalan (L-PAM). In this study, the mRNA levels of the different genes involved in NER (ERCC1, XPA, XPB, XPC, XPD, XPF) were examined in a panel of eight different human cancer cell lines, together with the overall DNA repair capacity using a host cell reactivation assay of a damaged plasmid. A statistically significant correlation was observed between the relative expression of XPA/XPC (P < 0.05) and ERCC1/XPC (P < 0.05) mRNAs. No correlation was found between the DDP and L-PAM IC50S and the relative mRNA expression of the tested NER genes. When the overall cellular DNA repair capacity was studied, carcinomas seemed to have a higher repair activity than leukaemias; but this repair DNA activity correlated neither with the mRNA expression of the different NER genes nor with DDP and L-PAM IC50S. These data seem to suggest that even if the NER pathway is an important determinant for the cytotoxicity of alkylating agents, as demonstrated by the extremely high sensitivity to alkylating agents in cells lacking this repair system, other factors have to play a role in regulating the cellular sensitivity/resistance to these antitumour drugs.

Interaction of MutS Protein with the Major and Minor Grooves of a Heteroduplex DNA

Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of Taq MutS protein to a heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24-28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish that Taq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1, 10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA.

Absence of UV-induced non-homologous recombination in repair-deficient CHO cell lines transfected with ERCC genes.

The nucleotide excision repair pathway removes a broad spectrum of DNA lesions, including UV-induced damage. To ascertain whether the repair of the latter has a causative role in the enhancement of non-homologous recombination, Chinese hamster CHO cell lines proficient and deficient in the ability to repair UV-induced damage were transfected with a plasmid containing the bacterial neoR gene. Following UV-treatment an enhancement of non-homologous recombination above the spontaneous level was observed in repair-proficient cells, whereas no increase was observed in repair-deficient cell lines. Hence, the latter were transfected with the corresponding excision repair cross complementing human genes and the resulting repair-proficient transfectants were tested for UV-induced non-homologous recombination. In both untreated and UV-treated transfectants, the frequencies of the event were not significantly different. Cumulatively, the results suggest that non-homologous recombination induced by UV-irradiation is not restored by the correction of the excision repair defect.

42 Deficiencies in DNA repair and its clinical impacts

All organisms have an evolved an intricate network of DNA repair systems, that permits elimination of virtually any type of DNA injury. Genetic defects in DNA repair pathways or in damage-induced cell cycle arrest result in genetic instability and a strong predisposition to cancer. Examples of this class of human syndromes are xeroderma pigmentosum (XP), Bloom's syndrome, Fanconi's anaemia, and ataxia telangiectasia. One of the major damage repair pathways is nucleotide excision repair (NER). This system. removes a broad spectrum of DNA lesions among which the mam UV-induced injury and bulky chemical adducts. Three NER-deficient human syndromes characterized by marked photosensitivity are known: XP, Cockayne syndrome (CS) and trichothiodystrophy (TTD). XP patients show pigmentation abnormalities and an over 1000 × increased risk of skin cancer caused by defects in one of at least 7 genes (XPA to XPG). CS displays sun sensitivity and overall developmental impairment. Two responsible genes are known: CSA and CSB. TTD is characterized by brittle hair, ichthyosis and many CS symptoms. The NER defect in TTD is due to mutations in TTDA, XPB or XPD. There are no indications for an increased risk of cancer in CS and TTD. Some patients show a combined XP + CS picture. They have been assigned to XPB, XPD or XPG. Several of the protein (complex)es involved in NER participate in other DNA transactions as well. The XPF/ERCC1 complex probably has a dual involvement in a mitotic recombination pathway. All three NER genes associated with TTD are also implicated in basal transcription. This notion has important clinical implications. It is likely that the TTD symptoms not explained by a NER defect are caused by subtle insufficiencies in basal transcription. Thus TTD may represent a combined ‘repair/transcription syndrome’. To analyze the complex geno-phenotype relationship between mutations in NER genes and the clinical symptoms we have utilized gene targeting in mouse embryonal stem cells, to generate mouse mutants carrying defined genetic changes in various repair genes. The mouse models provide valuable tools for studying relationship between molecular defect and clinical symptoms, including cancer predisposition. All organisms have an evolved an intricate network of DNA repair systems, that permits elimination of virtually any type of DNA injury. Genetic defects in DNA repair pathways or in damage-induced cell cycle arrest result in genetic instability and a strong predisposition to cancer. Examples of this class of human syndromes are xeroderma pigmentosum (XP), Bloom's syndrome, Fanconi's anaemia, and ataxia telangiectasia. One of the major damage repair pathways is nucleotide excision repair (NER). This system. removes a broad spectrum of DNA lesions among which the mam UV-induced injury and bulky chemical adducts. Three NER-deficient human syndromes characterized by marked photosensitivity are known: XP, Cockayne syndrome (CS) and trichothiodystrophy (TTD). XP patients show pigmentation abnormalities and an over 1000 × increased risk of skin cancer caused by defects in one of at least 7 genes (XPA to XPG). CS displays sun sensitivity and overall developmental impairment. Two responsible genes are known: CSA and CSB. TTD is characterized by brittle hair, ichthyosis and many CS symptoms. The NER defect in TTD is due to mutations in TTDA, XPB or XPD. There are no indications for an increased risk of cancer in CS and TTD. Some patients show a combined XP + CS picture. They have been assigned to XPB, XPD or XPG. Several of the protein (complex)es involved in NER participate in other DNA transactions as well. The XPF/ERCC1 complex probably has a dual involvement in a mitotic recombination pathway. All three NER genes associated with TTD are also implicated in basal transcription. This notion has important clinical implications. It is likely that the TTD symptoms not explained by a NER defect are caused by subtle insufficiencies in basal transcription. Thus TTD may represent a combined ‘repair/transcription syndrome’. To analyze the complex geno-phenotype relationship between mutations in NER genes and the clinical symptoms we have utilized gene targeting in mouse embryonal stem cells, to generate mouse mutants carrying defined genetic changes in various repair genes. The mouse models provide valuable tools for studying relationship between molecular defect and clinical symptoms, including cancer predisposition.

The actual incision determines the efficiency of repair of cisplatin-damaged DNA by the Escherichia coli UvrABC endonuclease.

The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.