Genome-wide analysis of transcription-coupled repair reveals novel transcription events in Caenorhabditis elegans.

Bulky DNA adducts such as those induced by ultraviolet light are removed from the genomes of multicellular organisms by nucleotide excision repair, which occurs through two distinct mechanisms, global repair, requiring the DNA damage recognition-factor XPC (xeroderma pigmentosum complementation group C), and transcription-coupled repair (TCR), which does not. TCR is initiated when elongating RNA polymerase II encounters DNA damage, and thus analysis of genome-wide excision repair in XPC-mutants only repairing by TCR provides a unique opportunity to map transcription events missed by methods dependent on capturing RNA transcription products and thus limited by their stability and/or modifications (5'-capping or 3'-polyadenylation). Here, we have performed eXcision Repair-sequencing (XR-seq) in the model organism Caenorhabditis elegans to generate genome-wide repair maps in a wild-type strain with normal excision repair, a strain lacking TCR (csb-1), and a strain that only repairs by TCR (xpc-1).

Cross-species investigation into the requirement of XPA for nucleotide excision repair.

After reconstitution of nucleotide excision repair (excision repair) with XPA, RPA, XPC, TFIIH, XPF-ERCC1 and XPG, it was concluded that these six factors are the minimal essential components of the excision repair machinery. All six factors are highly conserved across diverse organisms spanning yeast to humans, yet no identifiable homolog of the XPA gene exists in many eukaryotes including green plants. Nevertheless, excision repair is reported to be robust in the XPA-lacking organism, Arabidopsis thaliana, which raises a fundamental question of whether excision repair could occur without XPA in other organisms.

Genome-wide analysis of transcription-coupled repair reveals novel transcription events in .

Bulky DNA adducts such as those induced by ultraviolet light are removed from the genomes of multicellular organisms by nucleotide excision repair, which occurs through two distinct mechanisms, global repair, requiring the DNA damage recognition-factor XPC (xeroderma pigmentosum complementation group C), and transcription-coupled repair (TCR), which does not. TCR is initiated when elongating RNA polymerase II encounters DNA damage, and thus analysis of genome-wide excision repair in XPC-mutants only repairing by TCR provides a unique opportunity to map transcription events missed by methods dependent on capturing RNA transcription products and thus limited by their stability and/or modifications (5'-capping or 3'-polyadenylation). Here, we have performed the eXcision Repair-sequencing (XR-seq) in the model organism to generate genome-wide repair maps from a wild-type strain with normal excision repair, a strain lacking TCR (), or one that only repairs by TCR (-).

Nucleotide excision repair in Human cell lines lacking both XPC and CSB proteins.

Nucleotide excision repair removes UV-induced DNA damage through two distinct sub-pathways, global repair and transcription-coupled repair (TCR). Numerous studies have shown that in human and other mammalian cell lines that the XPC protein is required for repair of DNA damage from nontranscribed DNA via global repair and the CSB protein is required for repair of lesions from transcribed DNA via TCR. Therefore, it is generally assumed that abrogating both sub-pathways with an XPC-/-/CSB-/- double mutant would eliminate all nucleotide excision repair.

Molecular Mechanisms of Transcription-Coupled Repair.

Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including , yeast, , mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. , a notable exception, exhibits TCR but lacks an obvious TCR translocase.

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis.

In vitro and in vivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis.

Analysis of mammalian circadian clock protein complexes over a circadian cycle.

Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2, and PER3), CRY (CRY1 and CRY2), and CK1δ. In mice, peak expression of the repressors in the early morning reduces CLOCK- and BMAL1-mediated transcription/translation of the repressors themselves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24 h.

Effects of replication domains on genome-wide UV-induced DNA damage and repair.

Nucleotide excision repair is the primary repair mechanism that removes UV-induced DNA lesions in placentals. Unrepaired UV-induced lesions could result in mutations during DNA replication. Although the mutagenesis of pyrimidine dimers is reasonably well understood, the direct effects of replication fork progression on nucleotide excision repair are yet to be clarified.

Nucleotide excision repair removes thymidine analog 5-ethynyl-2'-deoxyuridine from the mammalian genome.

Nucleotide excision repair is the principal mechanism for removing bulky DNA adducts from the mammalian genome, including those induced by environmental carcinogens such as UV radiation, and anticancer drugs such as cisplatin. Surprisingly, we found that the widely used thymidine analog EdU is a substrate for excision repair when incorporated into the DNA of replicating cells. A number of thymidine analogs were tested, and only EdU was a substrate for excision repair.

Circadian clock, carcinogenesis, chronochemotherapy connections.

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers.

The Transcription-Repair Coupling Factor Mfd Prevents and Promotes Mutagenesis in a Context-Dependent Manner.

The (mutation frequency decline) gene was identified by screening an auxotrophic strain exposed to UV and held in a minimal medium before plating onto rich or minimal agar plates. It was found that, under these conditions, holding cells in minimal (nongrowth) conditions resulted in mutations that enabled cells to grow on minimal media. Using this observation as a starting point, a mutant was isolated that failed to mutate to auxotrophy under the prescribed conditions, and the gene responsible for this phenomenon (mutation frequency decline) was named .

Mycobacteria excise DNA damage in 12- or 13-nucleotide-long oligomers by prokaryotic-type dual incisions and performs transcription-coupled repair.

In nucleotide excision repair, bulky DNA lesions such as UV-induced cyclobutane pyrimidine dimers are removed from the genome by concerted dual incisions bracketing the lesion, followed by gap filling and ligation. So far, two dual-incision patterns have been discovered: the prokaryotic type, which removes the damage in 11-13-nucleotide-long oligomers, and the eukaryotic type, which removes the damage in 24-32-nucleotide-long oligomers. However, a recent study reported that the UvrC protein of removes damage in a manner analogous to yeast and humans in a 25-mer oligonucleotide arising from incisions at 15 nt from the 3´ end and 9 nt from the 5´ end flanking the damage.

Circadian regulation of c-MYC in mice.

The circadian clock is a global regulatory mechanism that controls the expression of 50 to 80% of transcripts in mammals. Some of the genes controlled by the circadian clock are oncogenes or tumor suppressors. Among these has been the focus of several studies which have investigated the effect of clock genes and proteins on transcription and MYC protein stability.

, which lacks canonical transcription-coupled repair proteins, performs transcription-coupled repair.

Previous work with the classic T4 endonuclease V digestion of DNA from irradiated cells followed by Southern hybridization led to the conclusion that lacks transcription-coupled repair (TCR). This conclusion was reinforced by the Genome Project, which revealed that lacks Cockayne syndrome WD repeat protein (CSA), CSB, or UV-stimulated scaffold protein A (UVSSA) homologs, whose orthologs are present in eukaryotes ranging from to humans that carry out TCR. A recently developed excision assay and the excision repair-sequencing (XR-Seq) method have enabled genome-wide analysis of nucleotide excision repair in various organisms at single-nucleotide resolution and in a strand-specific manner.

Mechanistic Study of TTF-1 Modulation of Cellular Sensitivity to Cisplatin.

The lung lineage master regulator gene, Thyroid Transcription Factor-1 (TTF-1, also known as NKX2-1), is used as a marker by pathologists to identify lung adenocarcinomas since TTF-1 is expressed in 60 ~ 70% of lung ADs. Much research has been conducted to investigate roles of TTF-1 in lung cancer biology. But, how it modulates cellular chemosensitivity remains poorly characterized.

Bringing It All Together: Coupling Excision Repair to the DNA Damage Checkpoint.

Nucleotide excision repair and the ATR-mediated DNA damage checkpoint are two critical cellular responses to the genotoxic stress induced by ultraviolet (UV) light and are important for cancer prevention. In vivo genetic data indicate that these global responses are coupled. Aziz Sancar et al.

Nucleotide excision repair by dual incisions in plants.

Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice.

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.

UV Light Potentiates STING (Stimulator of Interferon Genes)-dependent Innate Immune Signaling through Deregulation of ULK1 (Unc51-like Kinase 1).

The mechanism by which ultraviolet (UV) wavelengths of sunlight trigger or exacerbate the symptoms of the autoimmune disorder lupus erythematosus is not known but may involve a role for the innate immune system. Here we show that UV radiation potentiates STING (stimulator of interferon genes)-dependent activation of the immune signaling transcription factor interferon regulatory factor 3 (IRF3) in response to cytosolic DNA and cyclic dinucleotides in keratinocytes and other human cells. Furthermore, we find that modulation of this innate immune response also occurs with UV-mimetic chemical carcinogens and in a manner that is independent of DNA repair and several DNA damage and cell stress response signaling pathways.

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling.

The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1.

Circadian clock, cancer, and chemotherapy.

The circadian clock is a global regulatory system that interfaces with most other regulatory systems and pathways in mammalian organisms. Investigations of the circadian clock-DNA damage response connections have revealed that nucleotide excision repair, DNA damage checkpoints, and apoptosis are appreciably influenced by the clock. Although several epidemiological studies in humans and a limited number of genetic studies in mouse model systems have indicated that clock disruption may predispose mammals to cancer, well-controlled genetic studies in mice have not supported the commonly held view that circadian clock disruption is a cancer risk factor.

Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system.

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity.

Direct role for the replication protein treslin (Ticrr) in the ATR kinase-mediated checkpoint response.

TopBP1 (topoisomerase IIβ-binding protein 1) is a dual replication/checkpoint protein. Treslin/Ticrr, an essential replication protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclin E in a cell cycle-dependent manner, and its phosphorylation state dictates its interaction with TopBP1.