Jinchuan Hu

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.

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. In fact, in the appropriate genetic background, clock disruption may instead aid in cancer regression by promoting intrinsic and extrinsic apoptosis. Finally, the clock may affect the efficacy of cancer treatment (chronochemotherapy) by modulating the pharmacokinetics and pharmacodynamics of chemotherapeutic drugs as well as the activity of the DNA repair enzymes that repair the DNA damage caused by anticancer drugs.

Nucleotide Excision Repair in Human Cells FATE OF THE EXCISED OLIGONUCLEOTIDE CARRYING DNA DAMAGE IN VIVO

Background: Human excision repair removes UV photoproducts in 30-mers in vitro, but this has not been previously observed in vivo. Results: UV photoproducts are removed in vivo as 30-mers in complex with TFIIH both in general repair and in transcription-coupled repair. Conclusion: Primary products of excision repair have been isolated in vivo for the first time. Significance: The study provides novel insights into post-excision steps of human DNA repair. Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.

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

Significance 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. 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.

Highly specific and sensitive method for measuring nucleotide excision repair kinetics of ultraviolet photoproducts in human cells

The nucleotide excision repair pathway removes ultraviolet (UV) photoproducts from the human genome in the form of short oligonucleotides ∼30 nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The approach involves extraction of oligonucleotides from UV-irradiated cells, DNA end-labeling with biotin and streptavidin-mediated chemiluminescent detection of the excised UV photoproduct-containing oligonucleotides that are released from the genome during excision repair. Our novel approach is robust, with essentially no signal in the absence of UV or a functional excision repair system. Furthermore, our non-radioisotopic methodology allows for the sensitive detection of excision products within minutes following UV irradiation and does not require additional enrichment steps such as immunoprecipitation. Finally, this technique allows for quantitative measurements of excision repair in human cells. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.

DNA Repair Synthesis and Ligation Affect the Processing of Excised Oligonucleotides Generated by Human Nucleotide Excision Repair

Background: The mechanism of excised oligonucleotide processing during nucleotide excision repair is unknown. Results: UV photoproduct-containing oligonucleotides associate with chromatin following the dual incisions. Inhibition of gap-filling activities results in an accumulation of RPA-bound small, excised, damaged DNA (sedDNA) fragments. Conclusion: Gap filling-mediated dissociation of sedDNAs from RPA influences nucleotide excision repair rate. Significance: sedDNA processing is important in the DNA damage response. Ultraviolet (UV) photoproducts are removed from genomic DNA by dual incisions in humans in the form of 24- to 32-nucleotide-long oligomers (canonical 30-mers) by the nucleotide excision repair system. How the small, excised, damage-containing DNA oligonucleotides (sedDNAs) are processed in cells following the dual incision event is not known. Here, we demonstrate that sedDNAs are localized to the nucleus in two biochemically distinct forms, which include chromatin-associated, transcription factor II H-bound complexes and more readily solubilized, RPA-bound complexes. Because the nuclear mobility and repair functions of transcription factor II H and RPA are influenced by post-incision gap-filling events, we examined how DNA repair synthesis and DNA ligation affect sedDNA processing. We found that although these gap filling activities are not essential for the dual incision/sedDNA generation event per se, the inhibition of DNA repair synthesis and ligation is associated with a decrease in UV photoproduct removal rate and an accumulation of RPA-sedDNA complexes in the cell. These findings indicate that sedDNA processing and association with repair proteins following the dual incisions may be tightly coordinated with gap filling during nucleotide excision repair in vivo.

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

Significance 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. 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.

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

Significance 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. 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.

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

Significance 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. 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.

The Sac10b Homolog in Methanococcus maripaludis Binds DNA at Specific Sites

The Sac10b protein family, also known as Alba, is widely distributed in Archaea. Sac10b homologs in thermophilic Sulfolobus species are very abundant. They bind both DNA and RNA with high affinity and without sequence specificity, and their physiological functions are still not fully understood. Mma10b from the euryarchaeote Methanococcus maripaludis is a mesophilic member of the Sac10b family. Mma10b is not abundant and constitutes only approximately 0.01% of the total cellular protein. Disruption of mma10b resulted in poor growth of the mutant in minimal medium at near the optimal growth temperature but had no detectable effect on growth in rich medium. Quantitative proteomics, real time reverse transcription-PCR, and enzyme assays revealed that the expression levels of some genes involved in CO(2) assimilation and other activities were changed in the Deltamma10b mutant. Chromatin immunoprecipitation suggested a direct association of Mma10b with an 18-bp DNA binding motif in vivo. Electrophoretic mobility shift assays and DNase I footprinting confirmed that Mma10b preferentially binds specific sequences of DNA with an apparent Kd in the 100 nM range. These results suggested that the physiological role of Mma10b in the mesophilic methanococci is greatly diverged from that of homologs in thermophiles.