Involvement of nucleotide-excision repair in msh2 pms1-independent mismatch repair

Nucleotide-excision repair (NER) and mismatch repair (MMR) are prominent examples of highly conserved DNA repair systems which recognize and replace damaged and/or mispaired nucleotides in DNA. In humans, inheritable defects in components of the NER system are associated with severe diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS), whereas inactivation of MMR is accompanied by predisposition to certain types of cancer. In Schizosaccharomyces pombe, the msh2- and pms1-dependent long-patch MMR system efficiently corrects small insertion/deletion loops and all base-base mismatches, except C/C. Up to 70% of C/C mismatches generated in recombination intermediates, and to a lesser extent also other base-base mismatches, are thought to undergo correction by a minor, short-patch excision repair system. We identify here the NER genes rhpl4, swi10 and rad16 as components of this repair pathway and show that they act independently of msh2 and pms1.     

FAN1 mutations cause karyomegalic interstitial nephritis, linking chronic kidney failure to defective DNA damage repair

Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.     

Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis.

Double-strand DNA breaks (DSBs) pose a major threat to living cells, and several mechanisms for repairing these lesions have evolved. Eukaryotes can process DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ). NHEJ connects DNA ends irrespective of their sequence, and it predominates in mitotic cells, particularly during G1 (ref. 3). HR requires interaction of the broken DNA molecule with an intact homologous copy, and allows restoration of the original DNA sequence. HR is active during G2 of the mitotic cycle and predominates during meiosis, when the cell creates DSBs (ref. 4), which must be repaired by HR to ensure proper chromosome segregation. How the cell controls the choice between the two repair pathways is not understood. We demonstrate here a physical interaction between mammalian Ku70, which is essential for NHEJ (ref. 5), and Mre11, which functions both in NHEJ and meiotic HR (Refs 2,6). Moreover, we show that irradiated cells deficient for Ku70 are incapable of targeting Mre11 to subnuclear foci that may represent DNA-repair complexes. Nevertheless, Ku70 and Mre11 were differentially expressed during meiosis. In the mouse testis, Mre11 and Ku70 co-localized in nuclei of somatic cells and in the XY bivalent. In early meiotic prophase, however, when meiotic recombination is most probably initiated, Mre11 was abundant, whereas Ku70 was not detectable. We propose that Ku70 acts as a switch between the two DSB repair pathways. When present, Ku70 destines DSBs for NHEJ by binding to DNA ends and attracting other factors for NHEJ, including Mre11; when absent, it allows participation of DNA ends and Mre11 in the meiotic HR pathway.     

Germline epimutation of MLH1 in individuals with multiple cancers

Epigenetic silencing can mimic genetic mutation by abolishing expression of a gene. We hypothesized that an epimutation could occur in any gene as a germline event that predisposes to disease and looked for examples in tumor suppressor genes in individuals with cancer. Here we report two individuals with soma-wide, allele-specific and mosaic hypermethylation of the DNA mismatch repair gene MLH1. Both individuals lack evidence of genetic mutation in any mismatch repair gene but have had multiple primary tumors that show mismatch repair deficiency, and both meet clinical criteria for hereditary nonpolyposis colorectal cancer. The epimutation was also present in spermatozoa of one of the individuals, indicating a germline defect and the potential for transmission to offspring. Germline epimutation provides a mechanism for phenocopying of genetic disease. The mosaicism and nonmendelian inheritance that are characteristic of epigenetic states could produce patterns of disease risk that resemble those of polygenic or complex traits.     

Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia

The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.     

Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA

Genetic integrity is crucial to normal cell function, and mutations in genes required for DNA replication and repair underlie various forms of genetic instability and disease, including cancer. One structural feature of intact genomes is runs of homopolymeric dC/dG. Here we describe an unusual mutator phenotype in Caenorhabditis elegans characterized by deletions that start around the 3' end of polyguanine tracts and terminate at variable positions 5' from such tracts. We observed deletions throughout genomic DNA in about half of polyguanine tracts examined, especially those containing 22 or more consecutive guanine nucleotides. The mutator phenotype results from disruption of the predicted gene F33H2.1, which encodes a protein with characteristics of a DEAH helicase and which we have named dog-1 (for deletions of guanine-rich DNA). Nematodes mutated in dog-1 showed germline as well as somatic deletions in genes containing polyguanine tracts, such as vab-1. We propose that DOG-1 is required to resolve the secondary structures of guanine-rich DNA that occasionally form during lagging-strand DNA synthesis.     

Genome assembly comparison identifies structural variants in the human genome

Numerous types of DNA variation exist, ranging from SNPs to larger structural alterations such as copy number variants (CNVs) and inversions. Alignment of DNA sequence from different sources has been used to identify SNPs and intermediate-sized variants (ISVs). However, only a small proportion of total heterogeneity is characterized, and little is known of the characteristics of most smaller-sized (<50 kb) variants. Here we show that genome assembly comparison is a robust approach for identification of all classes of genetic variation. Through comparison of two human assemblies (Celera's R27c compilation and the Build 35 reference sequence), we identified megabases of sequence (in the form of 13,534 putative non-SNP events) that were absent, inverted or polymorphic in one assembly. Database comparison and laboratory experimentation further demonstrated overlap or validation for 240 variable regions and confirmed >1.5 million SNPs. Some differences were simple insertions and deletions, but in regions containing CNVs, segmental duplication and repetitive DNA, they were more complex. Our results uncover substantial undescribed variation in humans, highlighting the need for comprehensive annotation strategies to fully interpret genome scanning and personalized sequencing projects.     

Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation

Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential. The telomere also participates in processes of chromosomal repair, as evidenced by the 'capture' or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ). Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation. Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc-/- mice imparted a radiosensitivity syndrome associated with accelerated mortality. On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival. The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation. Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.     

The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin

The newly recognized ataxia-ocular apraxia 1 (AOA1; MIM 208920) is the most frequent cause of autosomal recessive ataxia in Japan and is second only to Friedreich ataxia in Portugal. It shares several neurological features with ataxia-telangiectasia, including early onset ataxia, oculomotor apraxia and cerebellar atrophy, but does not share its extraneurological features (immune deficiency, chromosomal instability and hypersensitivity to X-rays). AOA1 is also characterized by axonal motor neuropathy and the later decrease of serum albumin levels and elevation of total cholesterol. We have identified the gene causing AOA1 and the major Portuguese and Japanese mutations. This gene encodes a new, ubiquitously expressed protein that we named aprataxin. This protein is composed of three domains that share distant homology with the amino-terminal domain of polynucleotide kinase 3'- phosphatase (PNKP), with histidine-triad (HIT) proteins and with DNA-binding C2H2 zinc-finger proteins, respectively. PNKP is involved in DNA single-strand break repair (SSBR) following exposure to ionizing radiation and reactive oxygen species. Fragile-HIT proteins (FHIT) cleave diadenosine tetraphosphate, which is potentially produced during activation of the SSBR complex. The results suggest that aprataxin is a nuclear protein with a role in DNA repair reminiscent of the function of the protein defective in ataxia-telangiectasia, but that would cause a phenotype restricted to neurological signs when mutant.     

Genomic alterations in cultured human embryonic stem cells

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.     

Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss

DNA methyltransferase 1 (DNMT1) is crucial for maintenance of methylation, gene regulation and chromatin stability. DNA mismatch repair, cell cycle regulation in post-mitotic neurons and neurogenesis are influenced by DNA methylation. Here we show that mutations in DNMT1 cause both central and peripheral neurodegeneration in one form of hereditary sensory and autonomic neuropathy with dementia and hearing loss. Exome sequencing led to the identification of DNMT1 mutation c.1484A>G (p.Tyr495Cys) in two American kindreds and one Japanese kindred and a triple nucleotide change, c.1470-1472TCC>ATA (p.Asp490Glu-Pro491Tyr), in one European kindred. All mutations are within the targeting-sequence domain of DNMT1. These mutations cause premature degradation of mutant proteins, reduced methyltransferase activity and impaired heterochromatin binding during the G2 cell cycle phase leading to global hypomethylation and site-specific hypermethylation. Our study shows that DNMT1 mutations cause the aberrant methylation implicated in complex pathogenesis. The discovered DNMT1 mutations provide a new framework for the study of neurodegenerative diseases.     

The mismatch repair system is required for S-phase checkpoint activation

Defective S-phase checkpoint activation results in an inability to downregulate DNA replication following genotoxic insult such as exposure to ionizing radiation. This 'radioresistant DNA synthesis' (RDS) is a phenotypic hallmark of ataxia-telangiectasia, a cancer-prone disorder caused by mutations in ATM. The mismatch repair system principally corrects nucleotide mismatches that arise during replication. Here we show that the mismatch repair system is required for activation of the S-phase checkpoint in response to ionizing radiation. Cells deficient in mismatch repair proteins showed RDS, and restoration of mismatch repair function restored normal S-phase checkpoint function. Catalytic activation of ATM and ATM-mediated phosphorylation of the protein NBS1 (also called nibrin) occurred independently of mismatch repair. However, ATM-dependent phosphorylation and activation of the checkpoint kinase CHK2 and subsequent degradation of its downstream target, CDC25A, was abrogated in cells lacking mismatch repair. In vitro and in vivo approaches both show that MSH2 binds to CHK2 and that MLH1 associates with ATM. These findings indicate that the mismatch repair complex formed at the sites of DNA damage facilitates the phosphorylation of CHK2 by ATM, and that defects in this mechanism form the molecular basis for the RDS observed in cells deficient in mismatch repair.     

Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase eta

Oxidative damage to DNA has been proposed to have a role in cancer and ageing. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions. The ogg1Delta msh2Delta double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Delta or msh2Delta single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Poleta, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLeta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLeta in the yeast ogg1Delta mutant. Our results suggest an additional role for Poleta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.     

Cancer predisposition caused by elevated mitotic recombination in Bloom mice

Bloom syndrome is a disorder associated with genomic instability that causes affected people to be prone to cancer. Bloom cell lines show increased sister chromatid exchange, yet are proficient in the repair of various DNA lesions. The underlying cause of this disease are mutations in a gene encoding a RECQ DNA helicase. Using embryonic stem cell technology, we have generated viable Bloom mice that are prone to a wide variety of cancers. Cell lines from these mice show elevations in the rates of mitotic recombination. We demonstrate that the increased rate of loss of heterozygosity (LOH) resulting from mitotic recombination in vivo constitutes the underlying mechanism causing tumour susceptibility in these mice.     

Trinucleotide expansion in haploid germ cells by gap repair

Huntington disease (HD) is one of eight progressive neurodegenerative disorders in which the underlying mutation is a CAG expansion encoding a polyglutamine tract. The mechanism of trinucleotide expansion is poorly understood. Expansion is mediated by misaligned pairing of repeats and the inappropriate formation of DNA secondary structure as the duplex unpairs. It has never been clear, however, whether duplex unpairing occurs during mitotic replication or during strand-break repair. In simple organisms, trinucleotide expansion arises by replication slippage on either the leading or the lagging strand, homologous recombination, gene conversion, double-strand break repair and base excision repair; it is not clear which of these mechanisms is used in mammalian cells in vivo. We have followed heritable changes in CAG length in male transgenic mice. In germ cells, expansion is limited to the post-meiotic, haploid cell and therefore cannot involve mitotic replication or recombination between a homologous chromosome or a sister chromatid. Our data support a model in which expansion in the germ cells arises by gap repair and depends on a complex containing Msh2. Expansion occurs during gap-filling synthesis when DNA loops comprising the CAG trinucleotide repeats are sealed into the DNA strand.     

Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage

DNA damage surveillance networks in human cells can activate DNA repair, cell cycle checkpoints and apoptosis in response to fewer than four double-strand breaks (DSBs) per genome. These same networks tolerate telomeres, in part because the protein TRF2 prevents recognition of telomeric ends as DSBs by facilitating their organization into T loops. We now show that TRF2 associates with photo-induced DSBs in nontelomeric DNA in human fibroblasts within 2 s of irradiation. Unlike gammaH2AX, a common marker for DSB damage, TRF2 forms transient foci that colocalize closely with DSBs. The TRF2 DSB response requires the TRF2 basic domain but not its Myb domain and occurs in the absence of functional ATM and DNA-PK protein kinases, MRE11/Rad50/NBS1 complex and Ku70, WRN and BLM repair proteins. Furthermore, overexpression of TRF2 inhibits DSB-induced phosphorylation of ATM signaling targets. Our results implicate TRF2 in an initial stage of DSB recognition and processing that occurs before association of ATM with DSBs and activation of the ATM-dependent DSB response network.