Publications by authors named "Niels de Wind"

68 Publications

DNA mismatch repair-dependent DNA damage responses and cancer.

DNA Repair (Amst) 2020 09;93:102923

Department of Human Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands. Electronic address:

Canonical DNA mismatch repair (MMR) excises base-base mismatches to increase the fidelity of DNA replication. Thus, loss of MMR leads to increased spontaneous mutagenesis. MMR genes also are involved in the suppression of mutagenic, and the induction of protective, responses to various types of DNA damage. In this review we describe these non-canonical roles of MMR at different lesion types. Loss of non-canonical MMR gene functions may have important ramifications for the prevention, development and treatment of colorectal cancer associated with inherited MMR gene defects in Lynch syndrome. This graphical review pays tribute to Samuel H. Wilson. Sam not only made seminal contributions to understanding base excision repair, particularly with respect to structure-function relationships in DNA polymerase β but also, as Editor of DNA Repair, has maintained a high standard of the journal.
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http://dx.doi.org/10.1016/j.dnarep.2020.102923DOI Listing
September 2020

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation.

Front Genet 2020 27;11:798. Epub 2020 Jul 27.

Genetics and Computational Biology Department, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia.

Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants in , , and , including 12 missense variants that were also assessed using a cell-free MMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants.
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http://dx.doi.org/10.3389/fgene.2020.00798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398121PMC
July 2020

Digenic inheritance of MSH6 and MUTYH variants in familial colorectal cancer.

Genes Chromosomes Cancer 2020 Jul 2. Epub 2020 Jul 2.

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

We describe a family severely affected by colorectal cancer (CRC) where whole-exome sequencing identified the coinheritance of the germline variants encoding MSH6 p.Thr1100Met and MUTYH p.Tyr179Cys in, at least, three CRC patients diagnosed before 60 years of age. Digenic inheritance of monoallelic MSH6 variants of uncertain significance and MUTYH variants has been suggested to predispose to Lynch syndrome-associated cancers; however, cosegregation with disease in the familial setting has not yet been established. The identification of individuals carrying multiple potential cancer risk variants is expected to rise with the increased application of whole-genome sequencing and large multigene panel testing in clinical genetic counseling of familial cancer patients. Here we demonstrate the coinheritance of monoallelic variants in MSH6 and MUTYH consistent with cosegregation with CRC, further supporting a role for digenic inheritance in cancer predisposition.
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http://dx.doi.org/10.1002/gcc.22883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689793PMC
July 2020

Two integrated and highly predictive functional analysis-based procedures for the classification of MSH6 variants in Lynch syndrome.

Genet Med 2020 05 22;22(5):847-856. Epub 2020 Jan 22.

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Purpose: Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that gene. This not only obfuscates personalized health care but also the development of a rapid and reliable classification procedure that does not require clinical data.

Methods: The complete in vitro MMR activity (CIMRA) assay was calibrated against clinically classified MSH6 variants and, employing Bayes' rule, integrated with computational predictions of pathogenicity. To enable the validation of this two-component classification procedure we have employed a genetic screen to generate a large set of inactivating Msh6 variants, as proxies for pathogenic variants.

Results: The genetic screen-derived variants established that the two-component classification procedure displays high sensitivities and specificities. Moreover, these inactivating variants enabled the direct reclassification of human variants of uncertain significance (VUS) as (likely) pathogenic.

Conclusion: The two-component classification procedure and the genetic screens provide complementary approaches to rapidly and cost-effectively classify the large majority of human MSH6 variants. The approach followed here provides a template for the classification of variants in other disease-predisposing genes, facilitating the translation of personalized genomics into personalized health care.
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http://dx.doi.org/10.1038/s41436-019-0736-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200593PMC
May 2020

Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts.

DNA Repair (Amst) 2020 03 14;87:102771. Epub 2019 Dec 14.

Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan; Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka, 565-0871, Japan; National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan. Electronic address:

The (6-4) pyrimidine-pyrimidone photoproduct [(6-4)PP] is a major DNA lesion induced by ultraviolet radiation. (6-4)PP induces complex mutations opposite its downstream bases, in addition to opposite 3' or 5' base, as has been observed through a site-specific translesion DNA synthesis (TLS) assay. The mechanism by which these mutations occur is not well understood. To elucidate the mechanisms underlying mutagenesis induced by (6-4)PP, we performed an intracellular TLS assay using a replicative vector with site-specific T(thymidine)-T (6-4)PP. Rev3p53 mouse embryonic fibroblast (MEF) cells (defective in Polζ) were almost completely defective in bypassing T-T (6-4)PP, whereas both Rev1 and PolhPoliPolk MEF cells (defective in Polη, Polι, and Polκ) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Polζ plays an essential role, probably at the extension step. Among all cells tested, misincorporation occurred most frequently just beyond the lesion (position +1), indicating that the Polζ-dependent extension step is crucial for (6-4)PP-induced mutagenesis. We then examined the effects of sequence context on T-T (6-4)PP bypass using a series of T-T (6-4)PP templates with different sequences at position +1 or -1 to the lesion, and found that the dependency of T-T (6-4)PP bypass on Polζ is not sequence specific. However, the misincorporation frequency at position +1 differed significantly among these templates. The misincorporation of A at position +1 occurred frequently when a purine base was located at position -1. These results indicate that Polζ-dependent extension plays a major role in inducing base substitutions in (6-4)PP-induced mutagenesis, and its fidelity is affected by sequence context surrounding a lesion.
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http://dx.doi.org/10.1016/j.dnarep.2019.102771DOI Listing
March 2020

Mutagenic replication: target for tumor therapy?

Cell Res 2019 10;29(10):783-784

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

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http://dx.doi.org/10.1038/s41422-019-0218-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796867PMC
October 2019

A functional assay-based procedure to classify mismatch repair gene variants in Lynch syndrome.

Genet Med 2019 07 3;21(7):1486-1496. Epub 2018 Dec 3.

Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA.

Purpose: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data.

Methods: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories.

Results: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible.

Conclusion: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.
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http://dx.doi.org/10.1038/s41436-018-0372-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901556PMC
July 2019

Adjuvant Treatment for Proofreading Domain-Mutant Cancers: Sensitivity to Radiotherapy, Chemotherapy, and Nucleoside Analogues.

Clin Cancer Res 2018 07 20;24(13):3197-3203. Epub 2018 Mar 20.

Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands.

Pathogenic proofreading domain mutations are found in many malignancies where they are associated with ultramutation and favorable prognosis. The extent to which this prognosis depends on their sensitivity to adjuvant treatment is unknown, as is the optimal therapy for advanced-staged or recurrent -mutant cancers. We examined the recurrence-free survival of women with -mutant and wild-type endometrial cancers (EC) in the observation arm of the randomized PORTEC-1 endometrial cancer trial ( = 245 patients with stage I endometrial cancer for analysis). Sensitivity to radiotherapy and selected chemotherapeutics was compared between -mutant mouse-derived embryonic stem (mES) cells, generated using CRISPR-Cas9 ( mutations D275A/E275A, and cancer-associated P286R, S297F, V411L) and isogenic wild-type cell lines. In the observation arm of the PORTEC-1 trial ( = 245), women with -mutant endometrial cancers ( = 16) had an improved recurrence-free survival (10-year recurrence-free survival 100% vs. 80.1% for wild-type; HR, 0.143; 95% confidence interval, 0.001-0.996; = 0.049). mutations did not increase sensitivity to radiotherapy nor to chemotherapeutics in mES cells. In contrast, -mutant cells displayed significantly increased sensitivity to cytarabine and fludarabine (IC P286R-mutant vs. wild-type: 0.05 vs. 0.17 μmol/L for cytarabine, 4.62 vs. 11.1 μmol/L for fludarabine; < 0.001 for both comparisons). The favorable prognosis of -mutant cancers cannot be explained by increased sensitivity to currently used adjuvant treatments. These results support studies exploring minimization of adjuvant therapy for early-stage -mutant cancers, including endometrial and colorectal cancers. Conversely, mutations result in hypersensitivity to nucleoside analogues, suggesting the use of these compounds as a potentially effective targeted treatment for advanced-stage -mutant cancers. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0266DOI Listing
July 2018

Author Correction: Rev1 contributes to proper mitochondrial function via the PARP-NAD-SIRT1-PGC1α axis.

Sci Rep 2018 Mar 6;8(1):4245. Epub 2018 Mar 6.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-20586-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5840356PMC
March 2018

Rev1 contributes to proper mitochondrial function via the PARP-NAD-SIRT1-PGC1α axis.

Sci Rep 2017 10 2;7(1):12480. Epub 2017 Oct 2.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.

Nucleic acids, which constitute the genetic material of all organisms, are continuously exposed to endogenous and exogenous damaging agents, representing a significant challenge to genome stability and genome integrity over the life of a cell or organism. Unrepaired DNA lesions, such as single- and double-stranded DNA breaks (SSBs and DSBs), and single-stranded gaps can block progression of the DNA replication fork, causing replicative stress and/or cell cycle arrest. However, translesion synthesis (TLS) DNA polymerases, such as Rev1, have the ability to bypass some DNA lesions, which can circumvent the process leading to replication fork arrest and minimize replicative stress. Here, we show that Rev1-deficiency in mouse embryo fibroblasts or mouse liver tissue is associated with replicative stress and mitochondrial dysfunction. In addition, Rev1-deficiency is associated with high poly(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via the PARP-NAD-SIRT1-PGC1α axis.
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http://dx.doi.org/10.1038/s41598-017-12662-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624938PMC
October 2017

Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions.

Blood 2017 09 21;130(13):1523-1534. Epub 2017 Aug 21.

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for , a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of , essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.
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http://dx.doi.org/10.1182/blood-2017-01-764274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620415PMC
September 2017

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome.

Hum Mutat 2016 11 21;37(11):1162-1179. Epub 2016 Aug 21.

Department of Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands.

Monoallelic PMS2 germline mutations cause 5%-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA- and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.
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http://dx.doi.org/10.1002/humu.23052DOI Listing
November 2016

DNA mismatch repair: from biophysics to bedside.

DNA Repair (Amst) 2016 Feb 8;38:1-2. Epub 2015 Dec 8.

Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.dnarep.2015.11.007DOI Listing
February 2016

Post-translesion synthesis repair.

Oncotarget 2015 Aug;6(23):19342-3

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637279PMC
http://dx.doi.org/10.18632/oncotarget.4938DOI Listing
August 2015

FANCD2 and REV1 cooperate in the protection of nascent DNA strands in response to replication stress.

Nucleic Acids Res 2015 Sep 17;43(17):8325-39. Epub 2015 Jul 17.

Key Laboratory of Genomics and Precision Medicine, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China

REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.
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http://dx.doi.org/10.1093/nar/gkv737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787816PMC
September 2015

When mismatch repair met translesion synthesis.

Cell Cycle 2015 Aug 23;14(15):2377-8. Epub 2015 Jun 23.

a Department of Human Genetics ; Leiden University Medical Center ; Leiden , The Netherlands.

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http://dx.doi.org/10.1080/15384101.2015.1063288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613724PMC
August 2015

De novo mutations in PLXND1 and REV3L cause Möbius syndrome.

Nat Commun 2015 Jun 12;6:7199. Epub 2015 Jun 12.

Department of Human Genetics, Radboud University Medical Center, Donders Institute for Brain, Cognition and Behaviour, PO Box 9101, Nijmegen 6500 HB, The Netherlands.

Möbius syndrome (MBS) is a neurological disorder that is characterized by paralysis of the facial nerves and variable other congenital anomalies. The aetiology of this syndrome has been enigmatic since the initial descriptions by von Graefe in 1880 and by Möbius in 1888, and it has been debated for decades whether MBS has a genetic or a non-genetic aetiology. Here, we report de novo mutations affecting two genes, PLXND1 and REV3L in MBS patients. PLXND1 and REV3L represent totally unrelated pathways involved in hindbrain development: neural migration and DNA translesion synthesis, essential for the replication of endogenously damaged DNA, respectively. Interestingly, analysis of Plxnd1 and Rev3l mutant mice shows that disruption of these separate pathways converge at the facial branchiomotor nucleus, affecting either motoneuron migration or proliferation. The finding that PLXND1 and REV3L mutations are responsible for a proportion of MBS patients suggests that de novo mutations in other genes might account for other MBS patients.
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http://dx.doi.org/10.1038/ncomms8199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648025PMC
June 2015

Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions.

J Cell Biol 2015 Apr;209(1):33-46

Department of Human Genetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands

In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects.
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http://dx.doi.org/10.1083/jcb.201408017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395481PMC
April 2015

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease.

DNA Repair (Amst) 2015 May 21;29:56-64. Epub 2015 Jan 21.

Department of Human Genetics, Leiden University Medical Center, PO Box 9600, 2300RC Leiden, The Netherlands. Electronic address:

Most spontaneous and DNA damage-induced nucleotide substitutions in eukaryotes depend on translesion synthesis polymerases Rev1 and Pol ζ, the latter consisting of the catalytic subunit Rev3 and the accessory protein Rev7. Here we review the regulation, and the biochemical and cellular functions, of Rev1/Pol ζ-dependent translesion synthesis. These are correlated with phenotypes of mouse models with defects in Rev1, Rev3 or Rev7. The data indicate that Rev1/Pol ζ-mediated translesion synthesis is important for adaptive immunity while playing paradoxical roles in oncogenesis. On the other hand, by enabling the replication of endogenously damaged templates, Rev1/Pol ζ -dependent translesion synthesis protects stem cells, thereby preventing features of ageing. In conclusion, Rev1/Pol ζ-dependent translesion synthesis at DNA helix-distorting nucleotide lesions orchestrates pleiotropic responses that determine organismal fitness and disease.
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http://dx.doi.org/10.1016/j.dnarep.2015.01.001DOI Listing
May 2015

DNA lesion identity drives choice of damage tolerance pathway in murine cell chromosomes.

Nucleic Acids Res 2015 Feb;43(3):1637-45

DNA-damage tolerance (DDT) via translesion DNA synthesis (TLS) or homology-dependent repair (HDR) functions to bypass DNA lesions encountered during replication, and is critical for maintaining genome stability. Here, we present piggyBlock, a new chromosomal assay that, using piggyBac transposition of DNA containing a known lesion, measures the division of labor between the two DDT pathways. We show that in the absence of DNA damage response, tolerance of the most common sunlight-induced DNA lesion, TT-CPD, is achieved by TLS in mouse embryo fibroblasts. Meanwhile, BP-G, a major smoke-induced DNA lesion, is bypassed primarily by HDR, providing the first evidence for this mechanism being the main tolerance pathway for a biologically important lesion in a mammalian genome. We also show that, far from being a last-resort strategy as it is sometimes portrayed, TLS operates alongside nucleotide excision repair, handling 40% of TT-CPDs in repair-proficient cells. Finally, DDT acts in mouse embryonic stem cells, exhibiting the same pattern—mutagenic TLS included—despite the risk of propagating mutations along all cell lineages. The new method highlights the importance of HDR, and provides an effective tool for studying DDT in mammalian cells.
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http://dx.doi.org/10.1093/nar/gku1398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330363PMC
February 2015

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

Nucleic Acids Res 2015 Jan 10;43(1):282-94. Epub 2014 Dec 10.

Division of Biological Stress Responses, The Netherlands Cancer Institute, Amsterdam, The Netherlands

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.
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http://dx.doi.org/10.1093/nar/gku1301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288191PMC
January 2015

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light.

Nucleic Acids Res 2014 28;42(17):11071-82. Epub 2014 Aug 28.

Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6-4) pyrimidine pyrimidone photoproducts ((6-4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6-4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6-4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.
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http://dx.doi.org/10.1093/nar/gku779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176164PMC
February 2015

Maternal aldehyde elimination during pregnancy preserves the fetal genome.

Mol Cell 2014 Sep 21;55(6):807-817. Epub 2014 Aug 21.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; Department of Medicine, Level 5, Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 0QQ, UK. Electronic address:

Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2(-/-)Fanca(-/-) embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2.
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http://dx.doi.org/10.1016/j.molcel.2014.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175174PMC
September 2014

Functional implications of the p.Cys680Arg mutation in the MLH1 mismatch repair protein.

Mol Genet Genomic Med 2014 Jul 6;2(4):352-5. Epub 2014 May 6.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen DK-2200, Copenhagen N, Denmark.

In clinical genetic diagnostics, it is difficult to predict whether genetic mutations that do not greatly alter the primary sequence of the encoded protein causing unknown functional effects on cognate proteins lead to development of disease. Here, we report the clinical identification of c.2038 T>C missense mutation in exon 18 of the human MLH1 gene and biochemically characterization of the p.Cys680Arg mutant MLH1 protein to implicate it in the pathogenicity of the Lynch syndrome (LS). We show that the mutation is deficient in DNA mismatch repair and, therefore, contributing to LS in the carriers.
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http://dx.doi.org/10.1002/mgg3.80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113276PMC
July 2014

In memory of John Bruce Hays (1937-2014).

DNA Repair (Amst) 2014 Apr;16:vi-vii

Department of Biology, Stanford University, CA, United States.

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http://dx.doi.org/10.1016/S1568-7864(14)00071-8DOI Listing
April 2014

Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A.

Nucleic Acids Res 2014 Apr 23;42(7):4406-13. Epub 2014 Jan 23.

Department of Toxicogenetics, Leiden University Medical Center, PO box 9600, 2300 RC Leiden, The Netherlands.

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3(-/-) and Rev1(-/-) cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3(-/-) and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.
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http://dx.doi.org/10.1093/nar/gkt1412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985633PMC
April 2014

Consequences of germline variation disrupting the constitutional translational initiation codon start sites of MLH1 and BRCA2: Use of potential alternative start sites and implications for predicting variant pathogenicity.

Mol Carcinog 2015 Jul 2;54(7):513-22. Epub 2013 Dec 2.

Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However, few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site.
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http://dx.doi.org/10.1002/mc.22116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041856PMC
July 2015

Inactivation of DNA mismatch repair by variants of uncertain significance in the PMS2 gene.

Hum Mutat 2013 Nov 11;34(11):1477-80. Epub 2013 Sep 11.

Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands.

Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell-free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS.
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http://dx.doi.org/10.1002/humu.22426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858603PMC
November 2013

Rev1 is essential in generating G to C transversions downstream of the Ung2 pathway but not the Msh2+Ung2 hybrid pathway.

Eur J Immunol 2013 Oct 5;43(10):2765-70. Epub 2013 Aug 5.

Division of Biological Stress Response, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes are initiated by the enzymatic deamination of cytosine (C) to uracil (U). Uracil-DNA-glycosylase (Ung2) converts uracils into apyrimidinic (AP) sites, which is essential for the generation of transversions (TVs) at G/C basepairs during SHM and for efficient DNA break formation during CSR. Besides Ung2, the mismatch repair protein Msh2 and the translesion synthesis (TLS) DNA polymerase (Pol) Rev1 are implicated in SHM and CSR. To further unravel the role of Rev1, we studied WT, Rev1-deficient, Msh2-deficient, and Rev1, Msh2 double-deficient B cells. Loss of Rev1 only slightly reduced CSR. During SHM G/C to C/G TVs are generated in both Ung2- and Ung+Msh2-dependent fashions. We found that Rev1 is essential for the Msh2-independent generation of these TVs downstream of Ung2-induced AP sites. In the Ung+Msh2 hybrid pathway, Rev1 is not essential and can be substituted by an alternative TLS Pol, especially when Rev1 is lacking.
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http://dx.doi.org/10.1002/eji.201243191DOI Listing
October 2013

Genetic screens to identify pathogenic gene variants in the common cancer predisposition Lynch syndrome.

Proc Natl Acad Sci U S A 2013 Jun 20;110(23):9403-8. Epub 2013 May 20.

Department of Toxicogenetics, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands.

In many individuals suspected of the common cancer predisposition Lynch syndrome, variants of unclear significance (VUS), rather than an obviously pathogenic mutations, are identified in one of the DNA mismatch repair (MMR) genes. The uncertainty of whether such VUS inactivate MMR, and therefore are pathogenic, precludes targeted healthcare for both carriers and their relatives. To facilitate the identification of pathogenic VUS, we have developed an in cellulo genetic screen-based procedure for the large-scale mutagenization, identification, and cataloging of residues of MMR genes critical for MMR gene function. When a residue identified as mutated in an individual suspected of Lynch syndrome is listed as critical in such a reverse diagnosis catalog, there is a high probability that the corresponding human VUS is pathogenic. To investigate the applicability of this approach, we have generated and validated a prototypic reverse diagnosis catalog for the MMR gene MutS Homolog 2 (Msh2) by mutagenizing, identifying, and cataloging 26 deleterious mutations in 23 amino acids. Extensive in vivo and in vitro analysis of mutants listed in the catalog revealed both recessive and dominant-negative phenotypes. Nearly half of these critical residues match with VUS previously identified in individuals suspected of Lynch syndrome. This aids in the assignment of pathogenicity to these human VUS and validates the approach described here as a diagnostic tool. In a wider perspective, this work provides a model for the translation of personalized genomics into targeted healthcare.
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http://dx.doi.org/10.1073/pnas.1220537110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677471PMC
June 2013