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). Analysis of the intersections between the xpc-1 XR-seq repair maps with RNA-mapping datasets (RNA-seq, long- and short-capped RNA-seq) reveal previously unrecognized sites of transcription and further enhance our understanding of the genome of thtableis important model organism.

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 the eXcision Repair-sequencing (XR-seq) in the model organism Caenorhabditis elegans to generate genome-wide repair maps from a wild-type strain with normal excision repair, a strain lacking TCR (csb-1), or one that only repairs by TCR (xpc-1). Analysis of the intersections between the xpc-1 XR-seq repair maps with RNA-mapping datasets (RNA-seq, long- and short-capped RNA-seq) reveal previously unrecognized sites of transcription and further enhance our understanding of the genome of this important model organism.

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. Here, we performed a phylogenetic analysis of XPA across all species with annotated genomes and then quantitatively measured excision repair in the absence of XPA using the sensitive whole-genome qXR-Seq method in human cell lines and two model organisms, Caenorhabditis elegans and Drosophila melanogaster. We find that although the absence of XPA results in inefficient excision repair and UV-sensitivity in humans, flies, and worms, excision repair of UV-induced DNA damage is detectable over background. These studies have yielded a significant discovery regarding the evolution of XPA protein and its mechanistic role in nucleotide excision repair.

In vitro DNA repair genomics using XR-seq with Escherichia coli and mammalian cell-free extracts.

The XR-seq (eXcision Repair-sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell-free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light-irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell-free extracts and by mammalian cell-free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair.

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. Here we describe the construction of three different XPC-/-/CSB-/- human cell lines that, contrary to expectations, perform TCR. The XPC and CSB genes were mutated in cell lines derived from Xeroderma Pigmentosum patients as well as from normal human fibroblasts and repair was analyzed at the whole genome level using the very sensitive XR-seq method. As predicted, XPC-/- cells exhibited only TCR and CSB-/- cells exhibited only global repair. However, the XPC-/-/CSB-/- double mutant cell lines, although having greatly reduced repair, exhibited TCR. Mutating the CSA gene to generate a triple mutant XPC-/-/CSB-/-/CSA-/- cell line eliminated all residual TCR activity. Together, these findings provide new insights into the mechanistic features of mammalian nucleotide excision repair.

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. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E. coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

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. EdU excision was absent in repair-deficient cells, and in vitro, DNA duplexes bearing EdU were also substrates for excision by mammalian cell-free extracts. We used the excision repair sequencing (XR-seq) method to map EdU repair in the human genome at single-nucleotide resolution and observed that EdU was excised throughout the genome and was subject to transcription-coupled repair as evidenced by higher repair rates in the transcribed strand (TS) relative to the nontranscribed strand (NTS) in transcriptionally active genes. These properties of EdU, combined with its cellular toxicity and ability to cross the blood-brain barrier, make it a potential candidate for treating cancers of the brain, a tissue that typically demonstrates limited replication in adults.