Sheera Adar

Two‐polymerase mechanisms dictate error‐free and error‐prone translesion DNA synthesis in mammals

DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error‐free, and the third slow and error‐prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase ζ (polζ), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two‐polymerase combinations with polζ dictate error‐prone or error‐free TLS across the same lesion. These results highlight the central role of polζ in both error‐prone and error‐free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two‐polymerase combinations.

Quantitative measurement of translesion replication in human cells: Evidence for bypass of abasic sites by a replicative DNA polymerase

Mutations in oncogenes and tumor suppressor genes are critical in the development of cancer. A major pathway for the formation of mutations is the replication of unrepaired DNA lesions. To better understand the mechanism of translesion replication (TLR) in mammals, a quantitative assay for TLR in cultured cells was developed. The assay is based on the transient transfection of cultured cells with a gapped plasmid, carrying a site-specific lesion in the gap region. Filling in of the gap by TLR is assayed in a subsequent bioassay, by the ability of the plasmid extracted from the cells, to transform an Escherichia coli indicator strain. Using this method it was found that TLR through a synthetic abasic site in the adenocarcinoma H1299, the osteogenic sarcoma Saos-2, the prostate carcinoma PC3, and the hepatoma Hep3B cell lines occurred with efficiencies of 92 ± 6%, 32 ± 2%, 72 ± 4%, and 26 ± 3%, respectively. DNA sequence analysis showed that 85% of the bypass events in H1299 cells involved insertion of dAMP opposite the synthetic abasic site. Addition of aphidicolin, an inhibitor of DNA polymerases α, δ, and ɛ, caused a 4.4-fold inhibition of bypass. Analysis of two XP-V cell lines, defective in DNA polymerase η, showed bypass of 89%, indicating that polymerase η is not essential for bypass of abasic sites. These results suggest that in human cells bypass of abasic sites does not require the bypass-specific DNA polymerase η, but it does require at least one of the replicative DNA polymerases, α, δ, or ɛ. The quantitative TLR assay is expected to be useful in the molecular analysis of lesion bypass in a large variety of cultured mammalian 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.

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.

Repair of gaps opposite lesions by homologous recombination in mammalian cells

Damages in the DNA template inhibit the progression of replication, which may cause single-stranded gaps. Such situations can be tolerated by translesion DNA synthesis (TLS), or by homology-dependent repair (HDR), which is based on transfer or copying of the missing information from the replicated sister chromatid. Whereas it is well established that TLS plays an important role in DNA damage tolerance in mammalian cells, it is unknown whether HDR operates in this process. Using a newly developed plasmid-based assay that distinguishes between the three mechanisms of DNA damage tolerance, we found that mammalian cells can efficiently utilize HDR to repair DNA gaps opposite an abasic site or benzo[a]pyrene adduct. The majority of these events occurred by a physical strand transfer (homologous recombination repair; HRR), rather than a template switch mechanism. Furthermore, cells deficient in either the human RAD51 recombination protein or NBS1, but not Rad18, exhibited decreased gap repair through HDR, indicating a role for these proteins in DNA damage tolerance. To our knowledge, this is the first direct evidence of gap-lesion repair via HDR in mammalian cells, providing further molecular insight into the potential activity of HDR in overcoming replication obstacles and maintaining genome stability.

Gene Model 129 (Gm129) Encodes a Novel Transcriptional Repressor That Modulates Circadian Gene Expression

Background: Circadian gene expression in mammals is driven by rhythmic CLOCK·BMAL1 activities. Results: Gm129 binds to the CLOCK·BMAL1 complex on DNA and represses its transcriptional activator activity to regulate the circadian gene expression phase. Conclusion: Gm129 is a novel circadian clock modulator. Significance: The discovery of Gm129 reveals a new regulatory component of circadian gene expression. The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK·BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK·BMAL1·CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK·BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK·BMAL1 complex on DNA. Although Gm129−/− or Cry1−/− Gm129−/− mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit a significant phase delay compared with control. Our results suggest that, in addition to CRYs and PERs, the GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK·BMAL1 activity as a transcriptional repressor.

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.

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

Significance 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. 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.

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.

Erratum: Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals (The EMBO Journal (2009) 28 (992) DOI: 10.1038/emboj.2009.72)

U2OS cells were transfected with the indicated siRNAs. After incubation of 72 h, the plasmid mixtures containing the gap-lesion plasmid GP-BP-G1 or GP-cisPt-GG (kanR), along with the control gapped plasmid GP20 (cmR) and the carrier plasmid were introduced into the cells. After incubation of 8 h to allow TLS, the DNA was extracted and used to transform a recA E. coli indicator strain. GP-BP-G1 or GP-cisPt-GG (kanR) descendents were extracted from kanR colonies and subjected to DNA sequence analysis. Deletions and insertions are taken as non-TLS events. The number of clones, excluding clones having large deletions or insertions. The percent of mutagenic TLS is the fraction of events other than insertion of C opposite BP-G, or CC opposite cisPt-GG out of the total number of TLS events. P-value is given compared with the results obtained with cells transfected with control siRNA. Analysis was performed using the w test. P-values that have reached statistical significance are presented in bold type. The EMBO Journal (2009) 28, 992 | & 2009 European Molecular Biology Organization | All Rights Reserved 0261-4189/09 www.embojournal.org