Mutagenesis is a key hallmark and enabling characteristic of cancer cells, yet the diverse underlying mutagenic mechanisms that shape cancer genomes are not understood. This review will consider the ...emerging challenge of determining how DNA damage response pathways-both tolerance and repair-act upon specific forms of DNA damage to generate mutations characteristic of tumors. DNA polymerases are typically the ultimate mutagenic effectors of DNA repair pathways. Therefore, understanding the contributions of DNA polymerases is critical to develop a more comprehensive picture of mutagenic mechanisms in tumors. Selection of an appropriate DNA polymerase-whether error-free or error-prone-for a particular DNA template is critical to the maintenance of genome stability. We review different modes of DNA polymerase dysregulation including mutation, polymorphism, and over-expression of the polymerases themselves or their associated activators. Based upon recent findings connecting DNA polymerases with specific mechanisms of mutagenesis, we propose that compensation for DNA repair defects by error-prone polymerases may be a general paradigm molding the mutational landscape of cancer cells. Notably, we demonstrate that correlation of error-prone polymerase expression with mutation burden in a subset of patient tumors from The Cancer Genome Atlas can identify mechanistic hypotheses for further testing. We contrast experimental approaches from broad, genome-wide strategies to approaches with a narrower focus on a few hundred base pairs of DNA. In addition, we consider recent developments in computational annotation of patient tumor data to identify patterns of mutagenesis. Finally, we discuss the innovations and future experiments that will develop a more comprehensive portrait of mutagenic mechanisms in human tumors.
Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA ...Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.
Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)‐induced apoptosis and cell cycle arrest. ...DSBs may be invoked directly by ionizing radiation but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6‐4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently stalled and unresolved DNA replication forks can “collapse” to generate DSBs that induce signaling via Chk2 and its upstream activator the ataxia telangiectasia‐mutated (ATM) protein kinase. This review focuses on recently defined roles of Chk2 in protecting against DNA replication‐associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal “back‐up” roles for Chk2 in S‐phase progression and genomic stability. In humans, there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork‐stalling at estrogen‐DNA adducts and UV‐induced photolesions are implicated in the etiology of breast cancer and melanoma, respectively. It is likely therefore that the Chk2/CHEK2‐deficiency is associated with elevated risk for tumorigenesis caused by replication‐associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA‐damaging agents.
The Cancer/Testes (CT) Antigen HORMAD1 is germ cell-restricted and plays developmental roles in generation and processing of meiotic DNA Double Strand Breaks (DSB). Many tumors aberrantly overexpress ...HORMAD1 yet the potential impact of this CT antigen on cancer biology is unclear. We tested a potential role of HORMAD1 in genome maintenance in lung adenocarcinoma cells. We show that HORMAD1 re-distributes to nuclear foci and co-localizes with the DSB marker γH2AX in response to ionizing radiation (IR) and chemotherapeutic agents. The HORMA domain and C-term disordered oligomerization motif are necessary for localization of HORMAD1 to IR-induced foci (IRIF). HORMAD1-depleted cells are sensitive to IR and camptothecin. In reporter assays, Homologous Recombination (HR)-mediated repair of targeted ISce1-induced DSBs is attenuated in HORMAD1-depleted cells. In Non-Homologous End Joining (NHEJ) reporter assays, HORMAD1-depletion does not affect repair of ISce1-induced DSB. Early DSB signaling events (including ATM phosphorylation and formation of γH2AX, 53BP1 and NBS1 foci) are intact in HORMAD1-depleted cells. However, generation of RPA-ssDNA foci and redistribution of RAD51 to DSB are compromised in HORMAD1-depleted cells, suggesting that HORMAD1 promotes DSB resection. HORMAD1-mediated HR is a neomorphic activity that is independent of its meiotic partners (including HORMAD2 and CCDC36. Bioinformatic analysis of TCGA data show that similar to known HR pathway genes HORMAD1 is overexpressed in lung adenocarcinomas. Overexpression of HR genes is associated with specific mutational profiles (including copy number variation). Taken together, we identify HORMAD1-dependent DSB repair as a new mechanism of radioresistance and a probable determinant of mutability in lung adenocarcinoma.
KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; ...however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro. Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2–7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2–7 complex. KEAP1 is thus poised to affect MCM2–7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.
We have shown that smoking impacts bronchial airway gene expression and that heterogeneity in this response associates with smoking-related disease risk. In this study, we sought to determine whether ...microRNAs (miRNAs) play a role in regulating the airway gene expression response to smoking. We examined whole-genome miRNA and mRNA expression in bronchial airway epithelium from current and never smokers (n = 20) and found 28 miRNAs to be differentially expressed (P < 0.05) with the majority being down-regulated in smokers. We further identified a number of mRNAs whose expression level is highly inversely correlated with miRNA expression in vivo. Many of these mRNAs contain potential binding sites for the differentially expressed miRNAs in their 3'-untranslated region (UTR) and are themselves affected by smoking. We found that either increasing or decreasing the levels of mir-218 (a miRNA that is strongly affected by smoking) in both primary bronchial epithelial cells and H1299 cells was sufficient to cause a corresponding decrease or increase in the expression of predicted mir-218 mRNA targets, respectively. Further, mir-218 expression is reduced in primary bronchial epithelium exposed to cigarette smoke condensate (CSC), and alteration of mir-218 levels in these cells diminishes the induction of the predicted mir-218 target MAFG in response to CSC. These data indicate that mir-218 levels modulate the airway epithelial gene expression response to cigarette smoke and support a role for miRNAs in regulating host response to environmental toxins.
Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The ...genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides an aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O
mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.
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