The Role of the Rad55-Rad57 Complex in DNA Repair.

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55-Rad57 in HR.

Neuroblastoma Cells Depend on CSB for Faithful Execution of Cytokinesis and Survival.

Neuroblastoma, the most common extra-cranial solid tumor of early childhood, is one of the major therapeutic challenges in child oncology: it is highly heterogenic at a genetic, biological, and clinical level. The high-risk cases have one of the least favorable outcomes amongst pediatric tumors, and the mortality rate is still high, regardless of the use of intensive multimodality therapies. Here, we observed that neuroblastoma cells display an increased expression of Cockayne Syndrome group B (CSB), a pleiotropic protein involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division, and were recently found to confer cell robustness when they are up-regulated. In this study, we demonstrated that RNAi-mediated suppression of CSB drastically impairs tumorigenicity of neuroblastoma cells by hampering their proliferative, clonogenic, and invasive capabilities. In particular, we observed that CSB ablation induces cytokinesis failure, leading to caspases 9 and 3 activation and, subsequently, to massive apoptotic cell death. Worthy of note, a new frontier in cancer treatment, already proved to be successful, is cytokinesis-failure-induced cell death. In this context, CSB ablation seems to be a new and promising anticancer strategy for neuroblastoma therapy.

EXO1 resection at G-quadruplex structures facilitates resolution and replication.

G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruplexes, EXO1 resects the nascent DNA proximal to these structures to facilitate fork progression and faithful replication. In the absence of EXO1, forks accumulate at stabilized G-quadruplexes and ultimately collapse. These collapsed forks are preferentially repaired via error-prone end joining as depletion of EXO1 diverts repair away from error-free homology-dependent repair. Such aberrant repair leads to increased genomic instability, which is exacerbated at chromosome termini in the form of dysfunction and telomere loss.

New perspectives in cancer biology from a study of canonical and non-canonical functions of base excision repair proteins with a focus on early steps.

Alterations of DNA repair enzymes and consequential triggering of aberrant DNA damage response (DDR) pathways are thought to play a pivotal role in genomic instabilities associated with cancer development, and are further thought to be important predictive biomarkers for therapy using the synthetic lethality paradigm. However, novel unpredicted perspectives are emerging from the identification of several non-canonical roles of DNA repair enzymes, particularly in gene expression regulation, by different molecular mechanisms, such as (i) non-coding RNA regulation of tumour suppressors, (ii) epigenetic and transcriptional regulation of genes involved in genotoxic responses and (iii) paracrine effects of secreted DNA repair enzymes triggering the cell senescence phenotype. The base excision repair (BER) pathway, canonically involved in the repair of non-distorting DNA lesions generated by oxidative stress, ionising radiation, alkylation damage and spontaneous or enzymatic deamination of nucleotide bases, represents a paradigm for the multifaceted roles of complex DDR in human cells. This review will focus on what is known about the canonical and non-canonical functions of BER enzymes related to cancer development, highlighting novel opportunities to understand the biology of cancer and representing future perspectives for designing new anticancer strategies. We will specifically focus on APE1 as an example of a pleiotropic and multifunctional BER protein.

MutLγ promotes repeat expansion in a Fragile X mouse model while EXO1 is protective.

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases resulting from an expansion of a CGG-repeat tract at the 5' end of the FMR1 gene. The mechanism responsible for this unusual mutation is not fully understood. We have previously shown that mismatch repair (MMR) complexes, MSH2/MSH3 (MutSß) and MSH2/MSH6 (MutSα), together with Polß, a DNA polymerase important for base excision repair (BER), are important for expansions in a mouse model of these disorders. Here we show that MLH1/MLH3 (MutLγ), a protein complex that can act downstream of MutSß in MMR, is also required for all germ line and somatic expansions. However, exonuclease I (EXO1), which acts downstream of MutL proteins in MMR, is not required. In fact, a null mutation in Exo1 results in more extensive germ line and somatic expansions than is seen in Exo1+/+ animals. Furthermore, mice homozygous for a point mutation (D173A) in Exo1 that eliminates its nuclease activity but retains its native conformation, shows a level of expansion that is intermediate between Exo1+/+ and Exo1-/- animals. Thus, our data suggests that expansion of the FX repeat in this mouse model occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 protecting against expansion both in a nuclease-dependent and a nuclease-independent manner. Our data thus have implications for the expansion mechanism and add to our understanding of the genetic factors that may be modifiers of expansion risk in humans.

MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP.

Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool.

[Biomarkers of radiation-induced DNA repair processes]. / Radiothérapie et biomarqueurs de la réparation de l'ADN.

The identification of DNA repair biomarkers is of paramount importance. Indeed, it is the first step in the process of modulating radiosensitivity and radioresistance. Unlike tools of detection and measurement of DNA damage, DNA repair biomarkers highlight the variations of DNA damage responses, depending on the dose and the dose rate. The aim of the present review is to describe the main biomarkers of radiation-induced DNA repair. We will focus on double strand breaks (DSB), because of their major role in radiation-induced cell death. The most important DNA repair biomarkers are DNA damage signaling proteins, with ATM, DNA-PKcs, 53BP1 and γ-H2AX. They can be analyzed either using immunostaining, or using lived cell imaging. However, to date, these techniques are still time and money consuming. The development of "omics" technologies should lead the way to new (and usable in daily routine) DNA repair biomarkers.

MRE11 Promotes Tumorigenesis by Facilitating Resistance to Oncogene-Induced Replication Stress.

Hypomorphic mutations in the genes encoding the MRE11/RAD50/NBS1 (MRN) DNA repair complex lead to cancer-prone syndromes. MRN binds DNA double-strand breaks, where it functions in repair and triggers cell-cycle checkpoints via activation of the ataxia-telangiectasia mutated kinase. To gain understanding of MRN in cancer, we engineered mice with B lymphocytes lacking MRN, or harboring MRN in which MRE11 lacks nuclease activities. Both forms of MRN deficiency led to hallmarks of cancer, including oncogenic translocations involving c-Myc and the immunoglobulin locus. These preneoplastic B lymphocytes did not progress to detectable B lineage lymphoma, even in the absence of p53. Moreover, Mre11 deficiencies prevented tumorigenesis in a mouse model strongly predisposed to spontaneous B-cell lymphomas. Our findings indicate that MRN cannot be considered a standard tumor suppressor and instead imply that nuclease activities of MRE11 are required for oncogenesis. Inhibition of MRE11 nuclease activity increased DNA damage and selectively induced apoptosis in cells overexpressing oncogenes, suggesting MRE11 serves an important role in countering oncogene-induced replication stress. Thus, MRE11 may offer a target for cancer therapeutic development. More broadly, our work supports the idea that subtle enhancements of endogenous genome instability can exceed the tolerance of cancer cells and be exploited for therapeutic ends. Cancer Res; 77(19); 5327-38. ©2017 AACR.

Development of Damaged Nucleoside Mimics for Inhibition of Their Repair Enzymes.

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is a representative of nucleoside damage, which is generated by the reaction of the 8 position of dG with reactive oxygen species. Abundant 8-oxo-dG in DNA exhibits genotoxicity and has been linked to aging and disease, such as cancer. As the metabolism of cancer cells is much faster than that of normal cells, the oxidized product of the oligonucleotides and the nucleotide pool produces 8-oxo-dG and 8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), respectively. Human oxoguanine glycosylase (hOGG1) shows base excision activity for 8-oxo-dG in duplex DNA. On the other hand, human mutT homologue protein (hMTH1, also known as NUDT1) is important for oxidized nucleotide removal including 8-oxo-dGTP, and it is reported that the presence of hMTH1 is not essential for normal cells but is required for the survival of cancer cells. Therefore, we designed and synthesized 8-halogenated 7-deaza-2'-deoxyguanosine triphosphate (8-halo-7-deaza-dGTP) derivatives as mimics of 8-oxo-dGTP in order to interact with hMTH1. The 8-halo-7-deaza-dGTP derivatives were poor substrates for but strong binders to hMTH1. Interestingly, they exhibited strong competitive inhibition of hMTH1 in the hydrolysis of 8-oxo-dGTP. This inhibitory effect is caused by the slower rate of hydrolysis due to possible small enzyme structural changes. Although the detailed inhibition mechanism of the hydrolysis activity of hMTH1 is unknown, this result is the first to demonstrate the potential of nucleoside triphosphate derivatives as antitumor agents.

The Correlation of MGMT Promoter Methylation and Clinicopathological Features in Gastric Cancer: A Systematic Review and Meta-Analysis.

The silencing of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) by promoter methylation commonly occurs in human cancers. The relationship between MGMT promoter methylation and gastric cancer (GC) remains inconsistent. This study aimed to evaluate the potential value of MGMT promoter methylation in GC patients. Electronic databases were searched to identify eligible studies. The pooled odds ratio (OR) and the corresponding 95% confidence interval (95% CI) were used to evaluate the effects of MGMT methylation on GC risk and clinicopathological characteristics. In total, 31 eligible studies including 2988 GC patients and 2189 nonmalignant controls were involved in meta-analysis. In the pooled analysis, MGMT promoter methylation was significantly associated with GC risk (OR = 3.34, P < 0.001) and substantial heterogeneity (P < 0.001). Meta-regression and subgroup analyses based on the testing method, sample material and ethnicity failed to explain the sources of heterogeneity. Interestingly, MGMT methylation showed a trend associated with gender, and methylation is lower in males compared with females (OR = 0.76, 95% CI = 0.56-1.03). We did not find a significant association in relation to tumor types, clinical stage, age status or H. pylori status in cancer (all P > 0.1). MGMT promoter methylation may be correlated with the prognosis of GCs in disease free survival (DFS) or overall survival (OS) for univariate analysis. MGMT promoter methylation may play a crucial role in the carcinogenesis and prognosis of GC. MGMT methylation was not correlated with tumor types, clinical stage, age status, H. pylori status. However, the result of the association of MGMT methylation and gender should be considered with caution.

Scaffold attachment factor A (SAF-A) and Ku temporally regulate repair of radiation-induced clustered genome lesions.

Ionizing radiation (IR) induces highly cytotoxic double-strand breaks (DSBs) and also clustered oxidized bases in mammalian genomes. Base excision repair (BER) of bi-stranded oxidized bases could generate additional DSBs as repair intermediates in the vicinity of direct DSBs, leading to loss of DNA fragments. This could be avoided if DSB repair via DNA-PK-mediated nonhomologous end joining (NHEJ) precedes BER initiated by NEIL1 and other DNA glycosylases (DGs). Here we show that DNA-PK subunit Ku inhibits DGs via direct interaction. The scaffold attachment factor (SAF)-A, (also called hnRNP-U), phosphorylated at Ser59 by DNA-PK early after IR treatment, is linked to transient release of chromatin-bound NEIL1, thus preventing BER. SAF-A is subsequently dephosphorylated. Ku inhibition of DGs in vitro is relieved by unphosphorylated SAF-A, but not by the phosphomimetic Asp59 mutant. We thus propose that SAF-A, in concert with Ku, temporally regulates base damage repair in irradiated cell genome.

Mechanisms of tumor cell resistance to the current targeted-therapy agents.

Resistance to chemotherapy agents is a major challenge infront of cancer patient treatment and researchers. It is known that several factors, such as multidrug resistance proteins and ATP-binding cassette families, are cell membrane transporters that can efflux several substrates such as chemotherapy agents from the cell cytoplasm. To reduce the adverse effects of chemotherapy agents, various targeted-based cancer therapy (TBCT) agents have been developed. TBCT has revolutionized cancer treatment, and several agents have shown more specific effects on tumor cells than chemotherapies. Small molecule inhibitors and monoclonal antibodies are specific agents that mostly target tumor cells but have low side effects on normal cells. Although these agents have been very useful for cancer treatment, however, the presence of natural and acquired resistance has blunted the advantages of targeted therapies. Therefore, development of new options might be necessary. A better understanding of tumor cell resistance mechanisms to current treatment agents may provide an appropriate platform for developing and improving new treatment modalities. Therefore, in this review, different mechanisms of tumor cell resistance to chemotherapy drugs and current targeted therapies have been described.

EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele.

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.

Chemoresistance and chemotherapy targeting stem-like cells in malignant glioma.

Glioblastoma remains a tumor with a dismal prognosis because of failure of current treatment. Glioblastoma cells with stem cell (GSC) properties survive chemotherapy and give rise to tumor recurrences that invariably result in the death of the patients. Here we summarize the current knowledge on chemoresistance of malignant glioma with a strong focus on GSC. Chemoresistant GSC are the most likely cause of tumor recurrence, but it remains controversial if GSC and under which conditions GSC are more chemoresistant than non-GSC within the tumor. Regardless of this uncertainty, the chemoresistance varies and it is mainly mediated by intrinsic factors. O6-methyl-guanidine methyltransferase (MGMT) remains the most potent mediator of chemoresistance, but disturbed mismatch repair system and multidrug resistance proteins contribute substantially. However, the intrinsic resistance by MGMT expression is regulated by extrinsic factors like hypoxia increasing MGMT expression and thereby resistance to alkylating chemotherapy. The search of new biomarkers helping to predict the tumor response to chemotherapy is ongoing and will complement the already known markers like MGMT.

Akt-mediated phosphorylation of XLF impairs non-homologous end-joining DNA repair.

Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and non-homologous-end-joining (NHEJ). Although Akt has been reported to suppress HR, its role in NHEJ remains elusive. Here, we report that Akt phosphorylates XLF at Thr181 to trigger its dissociation from the DNA ligase IV/XRCC4 complex, and promotes its interaction with 14-3-3ß leading to XLF cytoplasmic retention, where cytosolic XLF is subsequently degraded by SCF(ß-TRCP) in a CKI-dependent manner. Physiologically, upon DNA damage, XLF-T181E expressing cells display impaired NHEJ and elevated cell death. Whereas a cancer-patient-derived XLF-R178Q mutant, deficient in XLF-T181 phosphorylation, exhibits an elevated tolerance of DNA damage. Together, our results reveal a pivotal role for Akt in suppressing NHEJ and highlight the tight connection between aberrant Akt hyper-activation and deficiency in timely DSB repair, leading to genomic instability and tumorigenesis.

Expression and clinical significance of P53, O6-methylguanine-dna methyltransferase and epidermal growth factor receptor in glioma.

Glioma is a serious life-threatening disease, the pathogenesis of which remains to be investigated. The objective of the present investigation was to explore the expression and clinical significance of tumor suppressor gene (P53), O6-methylguanine-DNA methyltransferase (MGMT) and epidermal growth factor receptor (EGFR) in glioma. Immunohistochemical staining was applied to study the clinical characteristics of 40 samples from glioma patients, detect the expression of and analyse the relationship between P53, MGMT and EGFR and glioma. The results demonstrated that the positive expression rate of P53 was 47.5% in 40 cases of glioma samples, of which the expression of P53 in the high grade glioma was higher than that of the low grade samples (P < 0.05); the positive expression rate of MGMT was 37.5%, but there was no significant significance of MGMT expression between the high grade glioma and the low grade glioma (P >0.05); the positive expression rate of EGFR was 55%, of which the expression of EGFR of the high grade glioma was higher than that of the low grade glioma (P<0.05). There was no significant difference in the expressions of P53, MGMT and EGFR in the glioma patients of different ages, gender and with different tumor sizes. The expressions of P53 and MGMT were negatively correlated (P<0.05). The expressions of P53 and EGFR were positively correlated (P<0.05). In conclusion, P53, EGFR and MGMT could play a role in the occurrence, development and deterioration of glioma.

Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

A role for XLF in DNA repair and recombination in human somatic cells.

Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor.

Characterizing DNA methylation alterations from The Cancer Genome Atlas.

The Cancer Genome Atlas (TCGA) Research Network is an ambitious multi-institutional consortium effort aimed at characterizing sequence, copy number, gene (mRNA) expression, microRNA expression, and DNA methylation alterations in 30 cancer types. TCGA data have become an extraordinary resource for basic, translational, and clinical researchers and have the potential to shape cancer diagnostic and treatment strategies. DNA methylation changes are integral to all aspects of cancer genomics and have been shown to have important associations with gene expression, sequence, and copy number changes. This Review highlights the knowledge gained from DNA methylation alterations in human cancers from TCGA.