The Cartography of UV-induced DNA Damage Formation and DNA Repair

DNA damage presents a barrier to DNA-templated biochemical processes, including gene expression and faithful DNA replication. Compromised DNA repair leads to mutations, enhancing the risk for genetic diseases and cancer development. Conventional experimental approaches to study DNA damage required a researcher to choose between measuring bulk damage over the entire genome, with little or no resolution regarding a specific location, and obtaining data specific to a locus of interest, without a global perspective. Recent advances in high-throughput genomic tools overcame these limitations and provide high-resolution measurements simultaneously across the genome. In this review, we discuss the available methods for measuring DNA damage and their repair, focusing on genomewide assays for pyrimidine photodimers, the major types of damage induced by ultraviolet irradiation. These new genomic assays will be a powerful tool in identifying key components of genome stability and carcinogenesis

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

Dynamic maps of UV damage formation and repair for the human genome

Nucleotide excision repair removes DNA damage caused by carcinogens, such as UV and anticancer drugs, such as cisplatin. We have developed two methods, high-sensitivity damage sequencing and excision repair sequencing that map the formation and repair of damage in the human genome at single-nucleotide resolution. The combination of dynamic damage and repair maps provides a holistic perspective of UV damage and repair of the human genome and has potential applications in cancer prevention and chemotherapy

Cisplatin DNA damage and repair maps of the human genome at single-nucleotide resolution

The chemotherapy drug cisplatin kills cancer cells by damaging their DNA. It has been used for treating a variety of cancer types for almost four decades. Although the drug is generally effective, it has strong adverse side effects, and some cancers exhibit or, after initial favorable response, develop drug resistance. The mechanism of drug resistance is multifactorial and involves the ability of cancer cells to repair the cisplatin-induced DNA damages. We have developed methods to map the sites of cisplatin damage and its repair for the entire human genome at single-nucleotide resolution. These methods can be used to study cancer sensitivity and resistance to the drugs, and to identify new strategies for efficient combination therapies

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis

Nucleotide excision repair is the sole mechanism for removing bulky adducts from the human genome, including those formed by UV radiation and chemotherapeutic drugs. We used eXcision Repair-sequencing, a genomic assay for measuring DNA repair, to map the kinetics of repair after UV treatment. These genome-wide repair maps, in turn, allowed us to infer how excision repair is influenced by DNA packaging. Active and open chromatin regions were repaired more rapidly than other genomic regions. Repair in repressed and heterochromatic regions is slower and persists for up to 2 d. Furthermore, late-repaired regions are associated with a higher level of cancer-linked somatic mutations, highlighting the importance of efficient DNA repair and linking chromatin organization to cancer mutagenesis

Genome-wide transcription-coupled repair in Escherichia coli is mediated by the Mfd translocase

In transcription-coupled repair (TCR), nucleotide excision repair occurs most rapidly in the template strand of actively transcribed genes. TCR has been observed in a limited set of genes directly assayed in Escherichia coli cells. In vitro, Mfd translocase performs reactions necessary to mediate TCR: It removes RNA polymerase blocked by a template strand lesion and rapidly delivers repair enzymes to the lesion. This study applied excision repair sequencing methodology to map the location of repair sites in different E. coli strains. Results showed that Mfd-dependent TCR is widespread in the E. coli genome. Results with UvrD helicase demonstrated its role in basal repair, but no overall role in TCR

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene

Benzo[a]pyrene (BaP) is a widespread potent carcinogen found in food, coal tar, cigarette smoke, and industrial smoke. Cigarette smoking is the leading cause of lung cancer, and the mutagenesis in smoking-associated lung cancer is determined by multiple factors, including nucleotide excision repair. We have developed a general method for genome-wide mapping of nucleotide excision repair at single-nucleotide resolution and applied it to generate repair maps of UV- and BaP-induced DNA damage in human. Results show a novel sequence specificity of BaP diol epoxide-deoxyguanosine repair. This general method can be used to study repair of all types of DNA damages that undergo nucleotide excision repair

Analysis of Ribonucleotide Removal from DNA by Human Nucleotide Excision Repair

Ribonucleotides are incorporated into the genome during DNA replication. The enzyme RNase H2 plays a critical role in targeting the removal of these ribonucleotides from DNA, and defects in RNase H2 activity are associated with both genomic instability and the human autoimmune/inflammatory disorder Aicardi-Goutières syndrome. Whether additional general DNA repair mechanisms contribute to ribonucleotide removal from DNA in human cells is not known. Because of its ability to act on a wide variety of substrates, we examined a potential role for canonical nucleotide excision repair in the removal of ribonucleotides from DNA. However, using highly sensitive dual incision/excision assays, we find that ribonucleotides are not efficiently targeted by the human nucleotide excision repair system in vitro or in cultured human cells. These results suggest that nucleotide excision repair is unlikely to play a major role in the cellular response to ribonucleotide incorporation in genomic DNA in human cells

Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution

We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine–pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells