Modulation of Cytotoxicity by Transcription-Coupled Nucleotide Excision Repair Is Independent of the Requirement for Bioactivation of Acylfulvene.

Bioactivation as well as DNA repair affects the susceptibility of cancer cells to the action of DNA-alkylating chemotherapeutic drugs. However, information is limited with regard to the relative contributions of these processes to the biological outcome of metabolically activated DNA alkylating agents. We evaluated the influence of cellular bioactivation capacity and DNA repair on cytotoxicity of the DNA alkylating agent acylfulvene (AF). We compared the cytotoxicity and RNA synthesis inhibition by AF and its synthetic activated analogue iso-M0 in a panel of fibroblast cell lines with deficiencies in transcription-coupled (TC-NER) or global genome nucleotide excision repair (GG-NER). We related these data to the inherent bioactivation capacity of each cell type on the basis of mRNA levels. We demonstrated that specific inactivation of TC-NER by siRNA had the largest positive impact on AF activity in a cancer cell line. These findings establish that transcription-coupled DNA repair reduces cellular sensitivity to AF, independent of the requirement for bioactivation.

Understanding photodermatoses associated with defective DNA repair: Syndromes with cancer predisposition.

Hereditary photodermatoses are a spectrum of rare photosensitive disorders that are often caused by genetic deficiency or malfunction of various components of the DNA repair pathway. This results clinically in extreme photosensitivity, with many syndromes exhibiting an increased risk of cutaneous malignancies. This review will focus specifically on the syndromes with malignant potential, including xeroderma pigmentosum, Bloom syndrome, and Rothmund-Thomson syndrome. The typical phenotypic findings of each disorder will be examined and contrasted, including noncutaneous identifiers to aid in diagnosis. The management of these patients will also be discussed. At this time, the mainstay of therapy remains strict photoprotection; however, genetic therapies are under investigation.

Understanding photodermatoses associated with defective DNA repair: Photosensitive syndromes without associated cancer predisposition.

Photodermatoses associated with defective DNA repair are a group of photosensitive hereditary skin disorders. In this review, we focus on diseases and syndromes with defective nucleotide excision repair that are not accompanied by an increased risk of cutaneous malignancies despite having photosensitivity. Specifically, the gene mutations and transcription defects, epidemiology, and clinical features of Cockayne syndrome, cerebro-oculo-facial-skeletal syndrome, ultraviolet-sensitive syndrome, and trichothiodystrophy will be discussed. These conditions may also have other extracutaneous involvement affecting the neurologic system and growth and development. Rigorous photoprotection remains an important component of the management of these inherited DNA repair-deficiency photodermatoses.

Transcription-coupled repair: an update.

Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. The serial steps in NER involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. Transcription-coupled repair (TCR) is a subpathway of NER dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, I report on recent findings that contribute to the elucidation of TCR mechanisms in the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae and human cells. I review general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.

Nucleotide excision repair in humans.

The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process.

Photosensitive human syndromes.

Photosensitivity in humans can result from defects in repair of light-induced DNA lesions, from photoactivation of chemicals (including certain medications) with sunlight to produce toxic mediators, and by immune reactions to sunlight exposures. Deficiencies in DNA repair and the processing of damaged DNA during replication and transcription may result in mutations and genomic instability. We will review current understanding of photosensitivity to short wavelength ultraviolet light (UV) due to genetic defects in particular DNA repair pathways; deficiencies in some are characterized by an extremely high incidence of cancer in sun-exposed tissues, while in others no cancers have been reported.

Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair.

New developments in comet-FISH.

The comet assay combined with fluorescence in-situ hybridisation (FISH) is a powerful technique for comparative analyses of damage induction and repair in genomes and in specific DNA sequences within single cells. Recent advances in the methodology of comet-FISH will be considered here, with particular attention to the design and generation of fluorescent probes. In general, all the approaches must fulfil a few basic requirements: the probes should be no longer than ~300 nucleotides in length (single or double stranded) to be able to penetrate the gel in which the target genomic DNA is embedded, they should be sequence-specific, and their signal should be detectable and distinct from the background fluorescence and the dye used to stain the DNA.

The complex choreography of transcription-coupled repair.

A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.

Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells.

Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

Transcription-coupled DNA repair in prokaryotes.

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that acts specifically on lesions in the transcribed strand of expressed genes. First reported in mammalian cells, TCR was then documented in Escherichia coli. In this organism, an RNA polymerase arrested at a lesion is displaced by the transcription repair coupling factor, Mfd. This protein recruits the NER lesion-recognition factor UvrA, and then dissociates from the DNA. UvrA binds UvrB, and the assembled UvrAB* complex initiates repair. In mutants lacking active Mfd, TCR is absent. A gene transcribed by the bacteriophage T7 RNA polymerase in E. coli also requires Mfd for TCR. The CSB protein (missing or defective in cells of patients with Cockayne syndrome, complementation group B) is essential for TCR in humans. CSB and its homologs in higher eukaryotes are likely functional equivalents of Mfd.

A novel XPD mutation in a compound heterozygote; the mutation in the second allele is present in three homozygous patients with mild sun sensitivity.

The XPD protein plays a pivotal role in basal transcription and in nucleotide excision repair (NER) as one of the ten known components of the transcription factor TFIIH. Mutations in XPD can result in the DNA repair-deficient diseases xeroderma pigmentosum (XP), trichothiodystrophy (TTD), cerebro-oculo-facial-skeletal syndrome, and in combined phenotypes such as XP/Cockayne syndrome and XP/TTD. We describe here an 18-year-old individual with mild sun sensitivity, no neurological abnormalities and no tumors, who carries a p.R683Q mutation in one allele, and the novel p.R616Q mutation in the other allele of the XPD gene. We also describe four patients from one family, homozygous for the identical p.R683Q mutation in XPD, who exhibit mild skin pigmentation and loss of tendon reflexes. Three homozygous patients presented with late-onset skin tumors, and two with features of premature aging and moderate cognitive decline. Cells from the compound heterozygous individual and from one of the patients homozygous for p.R683Q exhibited similar responses to UV irradiation: reduced viability and defective overall removal of UV-induced cyclobutane pyrimidine dimers, implying deficient global genomic NER. Cells from the compound heterozygous subject also failed to recover RNA synthesis after UV, indicating defective transcription-coupled NER. Mutations affecting codon 616 in XPD generally result in functionally null proteins; we hypothesize that the phenotype of the heterozygous patient results solely from expression of the p.R683Q allele. This study illustrates the importance of detailed follow up with sun sensitive individuals, to ensure appropriate prophylaxis and to understand the mechanistic basis of the implicated hereditary disease.

The Comet-FISH assay for the analysis of DNA damage and repair.

In this chapter, I describe the alkaline single-cell gel electrophoresis (Comet assay) combined with fluorescence in situ hybridization (FISH) technology, used in our laboratory, to study the incidence and repair of lesions induced in human cells by ultraviolet light. The Comet-FISH method permits the simultaneous and comparative analysis of DNA damage and its repair throughout the genome and in defined chromosomal regions. This very sensitive approach can be applied to any lesion, such as those induced by chemical carcinogens and products of cellular metabolism that can be converted to DNA single- or double-strand breaks. The unique advantages and limitations of the method for particular applications are discussed.

Hereditary photodermatoses.

Photodermatoses are defined as the abnormal reactions of the skin to photons, usually those of wavelengths found in sunlight. These reactions can be caused by a wide variety of reasons, including defects in repair oflight-induced DNA lesions, the interaction of certain chemicals or medications with sunlight to produce toxic mediators and photo-induced immune reactions. In this chapter we will describe photodermatoses that are associated with hereditary conditions. These can be subdivided into several groups: dermatoses caused by abnormal metabolic conditions, idiopathic photodermatoses, defects in cancer suppressor genes not directly involved in DNA repair but that predispose to photodistributed tumors and photosensitivity due to abnormalities in DNA repair pathways. Special emphasis will be placed on the relatively recently described UV-sensitive syndrome.

Impact of EMS outreach: successful developments in Latin America.

This collection of articles was inspired by the long-standing relationship between the Environmental Mutagen Society and Latin American scientists, and by the program for the 39th Environmental Mutagen Society meeting in Puerto Rico in 2008, which included a symposium featuring "South of the border" scientists. This collection, compiled by Graciela Spivak and Ofelia Olivero, both originally from Argentina, highlights scientists who work in or were trained in Latin American countries and in Puerto Rico in a variety of scientific specialties related to DNA repair and cancer susceptibility, genomic organization and stability, genetic diversity, and environmental contaminants.

A UV-sensitive syndrome patient with a specific CSA mutation reveals separable roles for CSA in response to UV and oxidative DNA damage.

UV-sensitive syndrome (UV(S)S) is a recently-identified autosomal recessive disorder characterized by mild cutaneous symptoms and defective transcription-coupled repair (TC-NER), the subpathway of nucleotide excision repair (NER) that rapidly removes damage that can block progression of the transcription machinery in actively-transcribed regions of DNA. Cockayne syndrome (CS) is another genetic disorder with sun sensitivity and defective TC-NER, caused by mutations in the CSA or CSB genes. The clinical hallmarks of CS include neurological/developmental abnormalities and premature aging. UV(S)S is genetically heterogeneous, in that it appears in individuals with mutations in CSB or in a still-unidentified gene. We report the identification of a UV(S)S patient (UV(S)S1VI) with a novel mutation in the CSA gene (p.trp361cys) that confers hypersensitivity to UV light, but not to inducers of oxidative damage that are notably cytotoxic in cells from CS patients. The defect in UV(S)S1VI cells is corrected by expression of the WT CSA gene. Expression of the p.trp361cys-mutated CSA cDNA increases the resistance of cells from a CS-A patient to oxidative stress, but does not correct their UV hypersensitivity. These findings imply that some mutations in the CSA gene may interfere with the TC-NER-dependent removal of UV-induced damage without affecting its role in the oxidative stress response. The differential sensitivity toward oxidative stress might explain the difference between the range and severity of symptoms in CS and the mild manifestations in UV(s)S patients that are limited to skin photosensitivity without precocious aging or neurodegeneration.

New applications of the Comet assay: Comet-FISH and transcription-coupled DNA repair.

Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30kb. The single cell gel electrophoresis (Comet) assay allows detection of single- or double-strand breaks at a 10-100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet "heads", and that Comet "tails" consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR.

Transcription-coupled DNA repair: two decades of progress and surprises.

Expressed genes are scanned by translocating RNA polymerases, which sensitively detect DNA damage and initiate transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes lesions from the template DNA strands of actively transcribed genes. Human hereditary diseases that present a deficiency only in TCR are characterized by sunlight sensitivity without enhanced skin cancer. Although multiple gene products are implicated in TCR, we still lack an understanding of the precise signals that can trigger this pathway. Futile cycles of TCR at naturally occurring non-canonical DNA structures might contribute to genomic instability and genetic disease.

In vivo assays for transcription-coupled repair.

This chapter describes the technologies used in our respective laboratories to study the incidence and repair of lesions induced in specific DNA sequences by ultraviolet light, chemical carcinogens, and products of cellular metabolism. The Southern blot method is suitable for analysis of damage and repair in the individual DNA strands of specific restriction fragments up to 25,000 nucleotides in length, whereas the ligation-mediated polymerase chain reaction approach permits analysis of shorter sequences at the nucleotide level. Both methods have unique advantages and limitations for particular applications.

Host cell reactivation of plasmids containing oxidative DNA lesions is defective in Cockayne syndrome but normal in UV-sensitive syndrome fibroblasts.

UV-sensitive syndrome (UV(S)S) is a human DNA repair-deficient disease with mild clinical manifestations. No neurological or developmental abnormalities or predisposition to cancer have been reported. In contrast, Cockayne syndrome (CS) patients exhibit severe developmental and neurological defects, in addition to photosensitivity. The cellular and biochemical responses of UV(S)S and CS cells to UV are indistinguishable, and result from defective transcription-coupled repair (TCR) of photoproducts in expressed genes. We propose that UV(S)S patients develop normally because they are proficient in repair of oxidative base damage. Consistent with our model, we show that Cockayne syndrome cells from complementation groups A and B (CS-A, CS-B) are more sensitive to treatment with hydrogen peroxide than wild type or UV(S)S cells. Using a host cell reactivation assay with plasmids containing UV-induced photoproducts, we find that expression of the plasmid-encoded lacZ gene is reduced in the TCR-deficient CS-B and UV(S)S cells. When the plasmids contain the oxidative base lesion thymine glycol, CS-B cells are defective in recovery of expression, whereas UV(S)S cells show levels of expression similar to those in wild type cells. 8-oxoguanine in the plasmids result in similarly defective host cell reactivation in CS-A and CS-B cells; abasic sites or single strand breaks in the plasmids cause similar decreases in expression in all the cell lines examined. Repair of thymine glycols in the lacZ gene was measured in plasmids extracted from transfected cells; removal of the lesions is efficient and without strand bias in all the cell lines tested.