Genome-wide mapping of protein-DNA damage interaction by PADD-seq.

Protein-DNA damage interactions are critical for understanding the mechanism of DNA repair and damage response. However, due to the relatively random distributions of UV-induced damage and other DNA bulky adducts, it is challenging to measure the interactions between proteins and these lesions across the genome. To address this issue, we developed a new method named Protein-Associated DNA Damage Sequencing (PADD-seq) that uses Damage-seq to detect damage distribution in chromatin immunoprecipitation-enriched DNA fragments. It is possible to delineate genome-wide protein-DNA damage interactions at base resolution with this strategy. Using PADD-seq, we observed that RNA polymerase II (Pol II) was blocked by UV-induced damage on template strands, and the interaction declined within 2 h in transcription-coupled repair-proficient cells. On the other hand, Pol II was clearly restrained at damage sites in the absence of the transcription-repair coupling factor CSB during the same time course. Furthermore, we used PADD-seq to examine local changes in H3 acetylation at lysine 9 (H3K9ac) around cisplatin-induced damage, demonstrating the method's broad utility. In conclusion, this new method provides a powerful tool for monitoring the dynamics of protein-DNA damage interaction at the genomic level, and it encourages comprehensive research into DNA repair and damage response.

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. Here, we applied Damage-seq and XR-seq techniques and generated replication maps in synchronized UV-treated HeLa cells. The results suggest that ongoing replication stimulates local repair in both early and late replication domains. Additionally, it was revealed that lesions on lagging strand templates are repaired slower in late replication domains, which is probably due to the imbalanced sequence context. Asymmetric relative repair is in line with the strand bias of melanoma mutations, suggesting a role of exogenous damage, repair, and replication in mutational strand asymmetry.

An analysis of health-related quality of life in children and adolescents after parotidectomy based on patient-reported outcomes.

PURPOSE: To assess health-related quality of life (HRQoL) and its influencing factors in these pediatric patients undergoing parotidectomy. METHODS: This was a cross-sectional study that included 37 children and adolescents (≤ 19 years) with parotid gland tumors who were treated in Sichuan Cancer Hospital between January 2006 and November 2021. HRQoL was assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 (EORTC QLQ-C30). The Wilcoxon rank sum test was used to analyze the factors influencing patients' HRQoL. RESULTS: 37 children and adolescents were included in the study, including 22 cases of benign tumors and 15 cases of malignant tumors. All patients underwent surgery, and some patients with malignant tumors received radiotherapy or chemotherapy. Malignancy, permanent facial palsy, and Frey syndrome were associated with worse HRQoL in children and adolescents with parotid gland tumors. Radiotherapy and no cervical lymph node dissection were associated with worse HRQoL in pediatric patients with malignancy. The surgical approach of parotid is not a factor influencing HRQoL. CONCLUSION: Factors associated with HRQoL in children and adolescents with parotid gland tumors include pathological types, permanent facial palsy, and Frey syndrome. In addition, factors affecting patients with malignancy include lateral lymph node dissection and radiotherapy.

A new technique for genome-wide mapping of nucleotide excision repair without immunopurification of damaged DNA.

Nucleotide excision repair functions to protect genome integrity, and ongoing studies using excision repair sequencing (XR-seq) have contributed to our understanding of how cells prioritize repair across the genome. In this method, the products of excision repair bearing damaged DNA are captured, sequenced, and then mapped genome-wide at single-nucleotide resolution. However, reagent requirements and complex procedures have limited widespread usage of this technique. In addition to the expense of these reagents, it has been hypothesized that the immunoprecipitation step using antibodies directed against damaged DNA may introduce bias in different sequence contexts. Here, we describe a newly developed adaptation called dA-tailing and adaptor ligation (ATL)-XR-seq, a relatively simple XR-seq method that avoids the use of immunoprecipitation targeting damaged DNA. ATL-XR-seq captures repair products by 3'-dA-tailing and 5'-adapter ligation instead of the original 5'- and 3'-dual adapter ligation. This new approach avoids adapter dimer formation during subsequent PCR, omits inefficient and time-consuming purification steps, and is very sensitive. In addition, poly(dA) tail length heterogeneity can serve as a molecular identifier, allowing more repair hotspots to be mapped. Importantly, a comparison of both repair mapping methods showed that no major bias is introduced by the anti-UV damage antibodies used in the original XR-seq procedure. Finally, we also coupled the described dA-tailing approach with quantitative PCR in a new method to quantify repair products. These new methods provide powerful and user-friendly tools to qualitatively and quantitatively measure excision repair.

Genome-wide analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine at single-nucleotide resolution unveils reduced occurrence of oxidative damage at G-quadruplex sites.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), one of the most common oxidative DNA damages, causes genome instability and is associated with cancer, neurological diseases and aging. In addition, OG and its repair intermediates can regulate gene transcription, and thus play a role in sensing cellular oxidative stress. However, the lack of methods to precisely map OG has hindered the study of its biological roles. Here, we developed a single-nucleotide resolution OG-sequencing method, named CLAPS-seq (Chemical Labeling And Polymerase Stalling Sequencing), to measure the genome-wide distribution of both exogenous and endogenous OGs with high specificity. Our data identified decreased OG occurrence at G-quadruplexes (G4s), in association with underrepresentation of OGs in promoters which have high GC content. Furthermore, we discovered that potential quadruplex sequences (PQSs) were hotspots of OGs, implying a role of non-G4-PQSs in OG-mediated oxidative stress response.

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.

UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution.

As an aberrant base in DNA, uracil is generated by either deoxyuridine (dU) misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Genome-wide profiles of uracil are important for study of these processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution, specificity, and/or sensitivity. Here, we developed a UdgX cross-linking and polymerase stalling sequencing ("Ucaps-seq") method to detect dU at single-nucleotide resolution. First, the specificity of Ucaps-seq was confirmed on synthetic DNA. Then the effectiveness of the approach was verified on two genomes from different sources. Ucaps-seq not only identified the enrichment of dU at dT sites in pemetrexed-treated cancer cells with globally elevated uracil but also detected dU at dC sites within the "WRC" motif in activated B cells which have increased dU in specific regions. Finally, Ucaps-seq was utilized to detect dU introduced by the cytosine base editor (nCas9-APOBEC) and identified a novel off-target site in cellular context. In conclusion, Ucaps-seq is a powerful tool with many potential applications, especially in evaluation of base editing fidelity.

Nucleotide excision repair: a versatile and smart toolkit.

Nucleotide excision repair (NER) is a major pathway to deal with bulky adducts induced by various environmental toxins in all cellular organisms. The two sub-pathways of NER, global genome repair (GGR) and transcription-coupled repair (TCR), differ in the damage recognition modes. In this review, we describe the molecular mechanism of NER in mammalian cells, especially the details of damage recognition steps in both sub-pathways. We also introduce new sequencing methods for genome-wide mapping of NER, as well as recent advances about NER in chromatin by these methods. Finally, the roles of NER factors in repairing oxidative damages and resolving R-loops are discussed.

Cisplatin-DNA adduct repair of transcribed genes is controlled by two circadian programs in mouse tissues.

Cisplatin is a major cancer chemotherapeutic drug. It kills cancer cells by damaging their DNA, mainly in the form of Pt-d(GpG) diadducts. However, it also has serious side effects, including nephrotoxicity and hepatotoxicity that limit its usefulness. Chronotherapy is taking circadian time into account during therapy to improve the therapeutic index, by improving efficacy and/or limiting toxicity. To this end, we tested the impact of clock time on excision repair of cisplatin-induced DNA damage at single-nucleotide resolution across the genome in mouse kidney and liver. We found that genome repair is controlled by two circadian programs. Repair of the transcribed strand (TS) of active, circadian-controlled genes is dictated by each gene's phase of transcription, which falls across the circadian cycle with prominent peaks at dawn and dusk. In contrast, repair of the nontranscribed strand (NTS) of all genes, repair of intergenic DNA, and global repair overall peaks at Zeitgeber time ZT08, as basal repair capacity, which is controlled by the circadian clock, peaks at this circadian time. Consequently, the TS and NTS of many genes are repaired out of phase. As most cancers are thought to have defective circadian rhythms, these results suggest that future research on timed dosage of cisplatin could potentially reduce damage to healthy tissue and improve its therapeutic index.

Single-nucleotide resolution analysis of nucleotide excision repair of ribosomal DNA in humans and mice.

The unique nucleolar environment, the repetitive nature of ribosomal DNA (rDNA), and especially the possible involvement of RNA polymerase I (RNAPI) in transcription-coupled repair (TCR) have made the study of repair of rDNA both interesting and challenging. TCR, the transcription-dependent, preferential excision repair of the template strand compared with the nontranscribed (coding) strand has been clearly demonstrated in genes transcribed by RNAPII. Whether TCR occurs in rDNA is unresolved. In the present work, we have applied analytical methods to map repair events in rDNA using data generated by the newly developed XR-seq procedure, which measures excision repair genome-wide with single-nucleotide resolution. We find that in human and mouse cell lines, rDNA is not subject to TCR of damage caused by UV or by cisplatin.

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

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

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

We used high-throughput sequencing of short, cyclobutane pyrimidine dimer-containing ssDNA oligos generated during repair of UV-induced damage to study that process at both mechanistic and systemic levels in Escherichia coli Numerous important insights on DNA repair were obtained, bringing clarity to the respective roles of UvrD helicase and Mfd translocase in repair of UV-induced damage. Mechanistically, experiments showed that the predominant role of UvrD in vivo is to unwind the excised 13-mer from dsDNA and that mutation of uvrD results in remarkable protection of that oligo from exonuclease activity as it remains hybridized to the dsDNA. Genome-wide analysis of the transcribed strand/nontranscribed strand (TS/NTS) repair ratio demonstrated that deletion of mfd globally shifts the distribution of TS/NTS ratios downward by a factor of about 2 on average for the most highly transcribed genes. Even for the least transcribed genes, Mfd played a role in preferential repair of the transcribed strand. On the other hand, mutation of uvrD, if anything, slightly pushed the distribution of TS/NTS ratios to higher ratios. These results indicate that Mfd is the transcription repair-coupling factor whereas UvrD plays a role in excision repair by aiding the catalytic turnover of excision repair proteins.

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

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is the major cause of lung cancer. BaP forms covalent DNA adducts after metabolic activation and induces mutations. We have developed a method for capturing oligonucleotides carrying bulky base adducts, including UV-induced cyclobutane pyrimidine dimers (CPDs) and BaP diol epoxide-deoxyguanosine (BPDE-dG), which are removed from the genome by nucleotide excision repair. The isolated oligonucleotides are ligated to adaptors, and after damage-specific immunoprecipitation, the adaptor-ligated oligonucleotides are converted to dsDNA with an appropriate translesion DNA synthesis (TLS) polymerase, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method translesion excision repair-sequencing (tXR-seq). In contrast to our previously described XR-seq method, tXR-seq does not depend on repair/removal of the damage in the excised oligonucleotides, and thus it is applicable to essentially all DNA damages processed by nucleotide excision repair. Here we present the excision repair maps for CPDs and BPDE-dG adducts generated by tXR-Seq for the human genome. In addition, we report the sequence specificity of BPDE-dG excision repair using tXR-seq.

RNA polymerase II is released from the DNA template during transcription-coupled repair in mammalian cells.

In mammalian cells, bulky DNA adducts located in the template but not the coding strand of genes block elongation by RNA polymerase II (RNAPII). The blocked RNAPII targets these transcription-blocking adducts to undergo more rapid excision repair than adducts located elsewhere in the genome. In excision repair, coupled incisions are made in the damaged DNA strand on both sides of the adduct. The fate of RNAPII in the course of this transcription-coupled repair (TCR) pathway is unclear. To address the fate of RNAPII, we used methods that control transcription to initiate a discrete "wave" of elongation complexes. Analyzing genome-wide transcription and repair by next-generation sequencing, we identified locations of elongation complexes and transcription-repair coupling events in genes throughout the genome. Using UV-exposed human skin fibroblasts, we found that, at the dose used, a single wave of elongation complexes was blocked within the first 25 kb of genes. TCR occurred where the elongation complexes were blocked, and repair was associated with the dissociation of these complexes. These results indicate that individual elongation complexes do not engage in multiple rounds of TCR with successive lesions. Our results are consistent with a model in which RNAPII is dissociated after the dual incision of the transcription-blocking lesion, perhaps by Cockayne syndrome group B translocase, or during the synthesis of a repair patch.

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

We recently developed a high-resolution genome-wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) and have now used XR-seq to determine which regions of the genome are subject to repair very soon after UV exposure and which regions are repaired later. Over a time course, we measured repair of the UV-induced damage of cyclobutane pyrimidine dimers (CPDs) (at 1, 4, 8, 16, 24, and 48 h) and (6-4)pyrimidine-pyrimidone photoproducts [(6-4)PPs] (at 5 and 20 min and 1, 2, and 4 h) in normal human skin fibroblasts. Each type of damage has distinct repair kinetics. The (6-4)PPs are detected as early as 5 min after UV treatment, with the bulk of repair completed by 4 h. Repair of CPDs, which we previously showed is intimately coupled to transcription, is slower and in certain regions persists even 2 d after UV irradiation. We compared our results to the Encyclopedia of DNA Elements data regarding histone modifications, chromatin state, and transcription. For both damage types, and for both transcription-coupled and general excision repair, the earliest repair occurred preferentially in active and open chromatin states. Conversely, repair in regions classified as "heterochromatic" and "repressed" was relatively low at early time points, with repair persisting into the late time points. Damage that remains during DNA replication increases the risk for mutagenesis. Indeed, late-repaired regions are associated with a higher level of cancer-linked mutations. In summary, we show that XR-seq is a powerful approach for studying relationships among chromatin state, DNA repair, genome stability, mutagenesis, and carcinogenesis.

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

Cisplatin is a major anticancer drug that kills cancer cells by damaging their DNA. Cancer cells cope with the drug by removal of the damages with nucleotide excision repair. We have developed methods to measure cisplatin adduct formation and its repair at single-nucleotide resolution. "Damage-seq" relies on the replication-blocking properties of the bulky base lesions to precisely map their location. "XR-seq" independently maps the removal of these damages by capturing and sequencing the excised oligomer released during repair. The damage and repair maps we generated reveal that damage distribution is essentially uniform and is dictated mostly by the underlying sequence. In contrast, cisplatin repair is heterogeneous in the genome and is affected by multiple factors including transcription and chromatin states. Thus, the overall effect of damages in the genome is primarily driven not by damage formation but by the repair efficiency. The combination of the Damage-seq and XR-seq methods has the potential for developing novel cancer therapeutic strategies.

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. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5' and 5 phosphodiester bonds 3' to the photoproduct.

Molecular mechanisms and genomic maps of DNA excision repair in Escherichia coli and humans.

Nucleotide excision repair is a major DNA repair mechanism in all cellular organisms. In this repair system, the DNA damage is removed by concerted dual incisions bracketing the damage and at a precise distance from the damage. Here, we review the basic mechanisms of excision repair in Escherichia coli and humans and the recent genome-wide mapping of DNA damage and repair in these organisms at single-nucleotide resolution.

Anomalous Capillary Rise under Nanoconfinement: A View of Molecular Kinetic Theory.

Understanding the capillary filling behaviors in nanopores is crucial for many science and engineering problems. Compared with the classical Bell-Cameron-Lucas-Washburn (BCLW) theory, anomalous coefficient is always observed because of the increasing role of surfaces. Here, a molecular kinetics approach is adopted to explain the mechanism of anomalous behaviors at the molecular level; a unified model taking account of the confined liquid properties (viscosity and density) and slip boundary condition is proposed to demonstrate the macroscopic consequences, and the model results are successfully validated against the published literature. The results show that (1) the effective viscosity induced by the interaction from the pore wall, as a function of wettability and the pore dimension (nanoslit height or nanotube diameter), may remarkably slow down the capillary filling process more than theoretically predicted. (2) The true slip, where water molecules directly slide on the walls, strongly depends on the wettability and will increase as the contact angle increases. In the hydrophilic nanopores, though, the magnitude may be comparable with the pore dimensions and promote the capillary filling compared with the classical BCLW model. (3) Compared with the other model, the proposed model can successfully predict the capillary filling for both faster or slower capillary filling process; meanwhile, it can capture the underlying physics behind these behaviors at the molecular level based on the effective viscosity and slippage. (4) The surface effects have different influence on the capillary filling in nanoslits and nanotubes, and the relative magnitude will change with the variation of wettability as well as the pore dimension.

Preparation and activity of glycosylated acetylsalicylic acid.

The glycosylated acetylsalicylic acid was prepared with bromo-α-d-galactose and acetylsalicylic acid. It indicated that the glycosylated acetylsalicylic acid had lower cytotoxicity than underivatized acetylsalicylic acid, and might selectively display anticancer activity in this situation that had enzyme or no enzyme.