TORC2 inhibition triggers yeast chromosome fragmentation through misregulated Base Excision Repair of clustered oxidation events.

Combinational therapies provoking cell death are of major interest in oncology. Combining TORC2 kinase inhibition with the radiomimetic drug Zeocin results in a rapid accumulation of double-strand breaks (DSB) in the budding yeast genome. This lethal Yeast Chromosome Shattering (YCS) requires conserved enzymes of base excision repair.

FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats.

FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding.

FANCD2-Associated Nuclease 1 Partially Compensates for the Lack of Exonuclease 1 in Mismatch Repair.

Germline mutations in the mismatch repair (MMR) genes , , , and are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in , encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1.

A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage.

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. Δ Δ Δ Δ Δ Δ Δ Δ and Δ produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications.

Transcription-coupled repair and mismatch repair contribute towards preserving genome integrity at mononucleotide repeat tracts.

The mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. Here, we introduce a method to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome.

ATAD5 deficiency alters DNA damage metabolism and sensitizes cells to PARP inhibition.

Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae).

Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding.

The enhancer/promoter of the vitellogenin II gene () has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation.

Synthetic lethality between BRCA1 deficiency and poly(ADP-ribose) polymerase inhibition is modulated by processing of endogenous oxidative DNA damage.

Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers.

Author Correction: FAN1 interaction with ubiquitylated PCNA alleviates replication stress and preserves genomic integrity independently of BRCA2.

The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following: This study was in part supported by the Swiss National Foundation Grant No.: 31003A-156023 to Alessandro Sartori.

FAN1 interaction with ubiquitylated PCNA alleviates replication stress and preserves genomic integrity independently of BRCA2.

Interstrand cross-link (ICL) hypersensitivity is a characteristic trait of Fanconi anemia (FA). Although FANCD2-associated nuclease 1 (FAN1) contributes to ICL repair, FAN1 mutations predispose to karyomegalic interstitial nephritis (KIN) and cancer rather than to FA. Thus, the biological role of FAN1 remains unclear.

Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair.

DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites.

Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination.

During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR.

FANCD2-associated nuclease 1, but not exonuclease 1 or flap endonuclease 1, is able to unhook DNA interstrand cross-links in vitro.

Cisplatin and its derivatives, nitrogen mustards and mitomycin C, are used widely in cancer chemotherapy. Their efficacy is linked primarily to their ability to generate DNA interstrand cross-links (ICLs), which effectively block the progression of transcription and replication machineries. Release of this block, referred to as unhooking, has been postulated to require endonucleases that incise one strand of the duplex on either side of the ICL.

Chromosome Missegregation Associated with RUVBL1 Deficiency.

RUVBL1 (RuvB-like1) and RUVBL2 (RuvB-like 2) are integral components of multisubunit protein complexes involved in processes ranging from cellular metabolism, transcription and chromatin remodeling to DNA repair. Here, we show that although RUVBL1 and RUVBL2 are known to form heterodimeric complexes in which they stabilize each other, the subunits separate during cytokinesis. In anaphase-to-telophase transition, RUVBL1 localizes to structures of the mitotic spindle apparatus, where it partially co-localizes with polo-like kinase 1 (PLK1).

Mismatch repair-dependent metabolism of O6-methylguanine-containing DNA in Xenopus laevis egg extracts.

The cytotoxicity of SN1-type alkylating agents such as N-methyl-N'-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O(6)-methylguanine ((me)G) in genomic DNA. One hypothesis posits that (me)G toxicity is caused by futile attempts of the mismatch repair (MMR) system to process (me)G/C or (me)G/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of (me)G-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways.

Influence of oxidized purine processing on strand directionality of mismatch repair.

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10(5)], and this precision is improved to about [1/10(7)] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information.

Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system.

Next-generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from rare variants remains daunting. We now describe an assay that permits the analysis of all types of mutations in any gene of choice through the generation of stable human cell lines, in which the endogenous protein has been inducibly replaced with its genetic variant.

High expression of the mismatch repair protein MSH6 is associated with poor patient survival in melanoma.

Objectives: The outcome of patients with primary melanoma (PM) cannot be completely explained based on currently adopted clinical-histopathologic criteria. In this study, we evaluated the potential prognostic value of mismatch repair protein expression in PMs.

Methods: We examined the immunohistochemical staining of mismatch repair proteins in 18 benign nevi and 101 stage I to III PMs and investigated their association with tumor clinicopathologic variables and melanoma mortality.

Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1.

Mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are associated with Lynch Syndrome (LS), a familial predisposition to early-onset cancer of the colon and other organs. Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome. This effort identified mutations in EXO1, which encodes the sole exonuclease implicated in MMR.

A comprehensive look at transcription factor gene expression changes in colorectal adenomas.

Background: Biological processes are controlled by transcription networks. Expression changes of transcription factor (TF) genes in precancerous lesions are therefore crucial events in tumorigenesis. Our aim was to obtain a comprehensive picture of these changes in colorectal adenomas.

Early insights into the function of KIAA1199, a markedly overexpressed protein in human colorectal tumors.

We previously reported that the expression of KIAA1199 in human colorectal tumors (benign and malignant) is markedly higher than that in the normal colonic mucosa. In this study, we investigated the functions of the protein encoded by this gene, which are thus far unknown. Immunostaining studies were used to reveal its subcellular localization, and proteomic and gene expression experiments were conducted to identify proteins that might interact with KIAA1199 and molecular pathways in which it might play roles.

Ribonucleotides misincorporated into DNA act as strand-discrimination signals in eukaryotic mismatch repair.

To improve replication fidelity, mismatch repair (MMR) must detect non-Watson-Crick base pairs and direct their repair to the nascent DNA strand. Eukaryotic MMR in vitro requires pre-existing strand discontinuities for initiation; consequently, it has been postulated that MMR in vivo initiates at Okazaki fragment termini in the lagging strand and at nicks generated in the leading strand by the mismatch-activated MLH1/PMS2 endonuclease. We now show that a single ribonucleotide in the vicinity of a mismatch can act as an initiation site for MMR in human cell extracts and that MMR activation in this system is dependent on RNase H2.

Postreplicative mismatch repair.

The mismatch repair (MMR) system detects non-Watson-Crick base pairs and strand misalignments arising during DNA replication and mediates their removal by catalyzing excision of the mispair-containing tract of nascent DNA and its error-free resynthesis. In this way, MMR improves the fidelity of replication by several orders of magnitude. It also addresses mispairs and strand misalignments arising during recombination and prevents synapses between nonidentical DNA sequences.