Publications by authors named "Richard D Kolodner"

126 Publications

Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae.

Nat Commun 2021 Sep 22;12(1):5568. Epub 2021 Sep 22.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0660, USA.

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways.
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http://dx.doi.org/10.1038/s41467-021-25866-zDOI Listing
September 2021

Ligation of newly replicated DNA controls the timing of DNA mismatch repair.

Curr Biol 2021 03 7;31(6):1268-1276.e6. Epub 2021 Jan 7.

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany; Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg 69120, Germany. Electronic address:

Mismatch repair (MMR) safeguards genome stability through recognition and excision of DNA replication errors. How eukaryotic MMR targets the newly replicated strand in vivo has not been established. MMR reactions reconstituted in vitro are directed to the strand containing a preexisting nick or gap, suggesting that strand discontinuities could act as discrimination signals. Another candidate is the proliferating cell nuclear antigen (PCNA) that is loaded at replication forks and is required for the activation of Mlh1-Pms1 endonuclease. Here, we discovered that overexpression of DNA ligase I (Cdc9) in Saccharomyces cerevisiae causes elevated mutation rates and increased chromatin-bound PCNA levels and accumulation of Pms1 foci that are MMR intermediates, suggesting that premature ligation of replication-associated nicks interferes with MMR. We showed that yeast Pms1 expression is mainly restricted to S phase, in agreement with the temporal coupling between MMR and DNA replication. Restricting Pms1 expression to the G2/M phase caused a mutator phenotype that was exacerbated in the absence of the exonuclease Exo1. This mutator phenotype was largely suppressed by increasing the lifetime of replication-associated DNA nicks, either by reducing or delaying Cdc9 ligase activity in vivo. Therefore, Cdc9 dictates a window of time for MMR determined by transient DNA nicks that direct the Mlh1-Pms1 in a strand-specific manner. Because DNA nicks occur on both newly synthesized leading and lagging strands, these results establish a general mechanism for targeting MMR to the newly synthesized DNA, thus preventing the accumulation of mutations that underlie the development of human cancer.
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http://dx.doi.org/10.1016/j.cub.2020.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8281387PMC
March 2021

FEN1 endonuclease as a therapeutic target for human cancers with defects in homologous recombination.

Proc Natl Acad Sci U S A 2020 08 27;117(32):19415-19424. Epub 2020 Jul 27.

Ludwig Institute for Cancer Research San Diego Branch, University of California San Diego School of Medicine, La Jolla, CA 92093-0669;

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show , which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed - and -defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from and mutations, and other genetic defects.
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http://dx.doi.org/10.1073/pnas.2009237117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431096PMC
August 2020

Tumour predisposition and cancer syndromes as models to study gene-environment interactions.

Nat Rev Cancer 2020 09 29;20(9):533-549. Epub 2020 May 29.

Thoracic Oncology, University of Hawaii Cancer Center, Honolulu, HI, USA.

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.
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http://dx.doi.org/10.1038/s41568-020-0265-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104546PMC
September 2020

Author Correction: Identification of Exo1-Msh2 interaction motifs in DNA mismatch repair and new Msh2-binding partners.

Nat Struct Mol Biol 2019 Dec;26(12):1184

Ludwig Institute for Cancer Research San Diego, University of California School of Medicine, San Diego, La Jolla, CA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41594-019-0337-4DOI Listing
December 2019

Essential genome instability suppressing genes identify potential human tumor suppressors.

Proc Natl Acad Sci U S A 2019 08 13;116(35):17377-17382. Epub 2019 Aug 13.

Ludwig Institute for Cancer Research, University of California San Diego School of Medicine, La Jolla, CA 92093-0669;

Gross Chromosomal Rearrangements (GCRs) play an important role in human diseases, including cancer. Although most of the nonessential Genome Instability Suppressing (GIS) genes in are known, the essential genes in which mutations can cause increased GCR rates are not well understood. Here 2 GCR assays were used to screen a targeted collection of temperature-sensitive mutants to identify mutations that caused increased GCR rates. This identified 94 essential GIS (eGIS) genes in which mutations cause increased GCR rates and 38 candidate eGIS genes that encode eGIS1 protein-interacting or family member proteins. Analysis of TCGA data using the human genes predicted to encode the proteins and protein complexes implicated by the eGIS genes revealed a significant enrichment of mutations affecting predicted human eGIS genes in 10 of the 16 cancers analyzed.
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http://dx.doi.org/10.1073/pnas.1906921116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717276PMC
August 2019

Alternative splicing regulates stochastic NLRP3 activity.

Nat Commun 2019 07 19;10(1):3238. Epub 2019 Jul 19.

Institute of Innate Immunity, University Hospital, University of Bonn, 53127, Bonn, Germany.

Leucine-rich repeat (LRR) domains are evolutionarily conserved in proteins that function in development and immunity. Here we report strict exonic modularity of LRR domains of several human gene families, which is a precondition for alternative splicing (AS). We provide evidence for AS of LRR domain within several Nod-like receptors, most prominently the inflammasome sensor NLRP3. Human NLRP3, but not mouse NLRP3, is expressed as two major isoforms, the full-length variant and a variant lacking exon 5. Moreover, NLRP3 AS is stochastically regulated, with NLRP3 ∆ exon 5 lacking the interaction surface for NEK7 and hence loss of activity. Our data thus reveals unexpected regulatory roles of AS through differential utilization of LRRs modules in vertebrate innate immunity.
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http://dx.doi.org/10.1038/s41467-019-11076-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642158PMC
July 2019

Inhibition of Nuclear PTEN Tyrosine Phosphorylation Enhances Glioma Radiation Sensitivity through Attenuated DNA Repair.

Cancer Cell 2019 03 28;35(3):504-518.e7. Epub 2019 Feb 28.

Ludwig Institute for Cancer Research, San Diego Branch, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA; Department of Neurosurgery, University of Minnesota, Minneapolis, MN 55455, USA; Department of Pathology, School of Medicine, University of California at San Diego, La Jolla, CA 92093, USA. Electronic address:

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.
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http://dx.doi.org/10.1016/j.ccell.2019.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424615PMC
March 2019

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Microb Cell 2019 Jan 7;6(1):1-64. Epub 2019 Jan 7.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Durham, NC, USA.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
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http://dx.doi.org/10.15698/mic2019.01.664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334234PMC
January 2019

The properties of Msh2-Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair.

J Biol Chem 2018 11 20;293(47):18055-18070. Epub 2018 Sep 20.

From the Ludwig Institute for Cancer Research San Diego,; Cellular and Molecular Medicine,; Moores-UCSD Cancer Center, and; Institute of Genomic Medicine, University of California School of Medicine, San Diego, La Jolla, California 92093-0669. Electronic address:

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-Pms1-independent and Mlh1-Pms1-dependent MMR reactions and in activation of the Mlh1-Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.
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http://dx.doi.org/10.1074/jbc.RA118.005439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6254361PMC
November 2018

The Swr1 chromatin-remodeling complex prevents genome instability induced by replication fork progression defects.

Nat Commun 2018 09 11;9(1):3680. Epub 2018 Sep 11.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0669, USA.

Genome instability is associated with tumorigenesis. Here, we identify a role for the histone Htz1, which is deposited by the Swr1 chromatin-remodeling complex (SWR-C), in preventing genome instability in the absence of the replication fork/replication checkpoint proteins Mrc1, Csm3, or Tof1. When combined with deletion of SWR1 or HTZ1, deletion of MRC1, CSM3, or TOF1 or a replication-defective mrc1 mutation causes synergistic increases in gross chromosomal rearrangement (GCR) rates, accumulation of a broad spectrum of GCRs, and hypersensitivity to replication stress. The double mutants have severe replication defects and accumulate aberrant replication intermediates. None of the individual mutations cause large increases in GCR rates; however, defects in MRC1, CSM3 or TOF1 cause activation of the DNA damage checkpoint and replication defects. We propose a model in which Htz1 deposition and retention in chromatin prevents transiently stalled replication forks that occur in mrc1, tof1, or csm3 mutants from being converted to DNA double-strand breaks that trigger genome instability.
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http://dx.doi.org/10.1038/s41467-018-06131-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134005PMC
September 2018

Identification of Exo1-Msh2 interaction motifs in DNA mismatch repair and new Msh2-binding partners.

Nat Struct Mol Biol 2018 08 30;25(8):650-659. Epub 2018 Jul 30.

Ludwig Institute for Cancer Research San Diego, University of California School of Medicine, San Diego, La Jolla, CA, USA.

Eukaryotic DNA mismatch repair (MMR) involves both exonuclease 1 (Exo1)-dependent and Exo1-independent pathways. We found that the unstructured C-terminal domain of Saccharomyces cerevisiae Exo1 contains two MutS homolog 2 (Msh2)-interacting peptide (SHIP) boxes downstream from the MutL homolog 1 (Mlh1)-interacting peptide (MIP) box. These three sites were redundant in Exo1-dependent MMR in vivo and could be replaced by a fusion protein between an N-terminal fragment of Exo1 and Msh6. The SHIP-Msh2 interactions were eliminated by the msh2 mutation, and wild-type but not mutant SHIP peptides eliminated Exo1-dependent MMR in vitro. We identified two S. cerevisiae SHIP-box-containing proteins and three candidate human SHIP-box-containing proteins. One of these, Fun30, had a small role in Exo1-dependent MMR in vivo. The Remodeling of the Structure of Chromatin (Rsc) complex also functioned in both Exo1-dependent and Exo1-independent MMR in vivo. Our results identified two modes of Exo1 recruitment and a peptide module that mediates interactions between Msh2 and other proteins, and they support a model in which Exo1 functions in MMR by being tethered to the Msh2-Msh6 complex.
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http://dx.doi.org/10.1038/s41594-018-0092-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837739PMC
August 2018

SUMO E3 ligase Mms21 prevents spontaneous DNA damage induced genome rearrangements.

PLoS Genet 2018 03 5;14(3):e1007250. Epub 2018 Mar 5.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO). Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs) selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.
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http://dx.doi.org/10.1371/journal.pgen.1007250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860785PMC
March 2018

Cdc73 suppresses genome instability by mediating telomere homeostasis.

PLoS Genet 2018 01 10;14(1):e1007170. Epub 2018 Jan 10.

Ludwig Institute for Cancer Research, San Diego Branch, San Diego, California, United States of America.

Defects in the genes encoding the Paf1 complex can cause increased genome instability. Loss of Paf1, Cdc73, and Ctr9, but not Rtf1 or Leo1, caused increased accumulation of gross chromosomal rearrangements (GCRs). Combining the cdc73Δ mutation with individual deletions of 43 other genes, including TEL1 and YKU80, which are involved in telomere maintenance, resulted in synergistic increases in GCR rates. Whole genome sequence analysis of GCRs indicated that there were reduced relative rates of GCRs mediated by de novo telomere additions and increased rates of translocations and inverted duplications in cdc73Δ single and double mutants. Analysis of telomere lengths and telomeric gene silencing in strains containing different combinations of cdc73Δ, tel1Δ and yku80Δ mutations suggested that combinations of these mutations caused increased defects in telomere maintenance. A deletion analysis of Cdc73 revealed that a central 105 amino acid region was necessary and sufficient for suppressing the defects observed in cdc73Δ strains; this region was required for the binding of Cdc73 to the Paf1 complex through Ctr9 and for nuclear localization of Cdc73. Taken together, these data suggest that the increased GCR rate of cdc73Δ single and double mutants is due to partial telomere dysfunction and that Ctr9 and Paf1 play a central role in the Paf1 complex potentially by scaffolding the Paf1 complex subunits or by mediating recruitment of the Paf1 complex to the different processes it functions in.
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http://dx.doi.org/10.1371/journal.pgen.1007170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5779705PMC
January 2018

Analyzing Genome Rearrangements in Saccharomyces cerevisiae.

Methods Mol Biol 2018 ;1672:43-61

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0669, USA.

Genome rearrangements underlie different human diseases including many cancers. Determining the rates at which genome rearrangements arise and isolating unique, independent genome rearrangements is critical to understanding the genes and pathways that prevent or promote genome rearrangements. Here, we describe quantitative S. cerevisiae genetic assays for measuring the rates of accumulating genome rearrangements including deletions, translocations, and broken chromosomes healed by de novo telomere addition that result in the deletion of two counter-selectable genes, CAN1 and URA3, placed in the nonessential regions of the S. cerevisiae genome. The assays also allow for the isolation of individual genome rearrangements for structural studies, and a method for analyzing genome rearrangements by next-generation DNA sequencing is provided.
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http://dx.doi.org/10.1007/978-1-4939-7306-4_5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657460PMC
June 2018

Pathways and Mechanisms that Prevent Genome Instability in .

Genetics 2017 07;206(3):1187-1225

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California 92093-0669

Genome rearrangements result in mutations that underlie many human diseases, and ongoing genome instability likely contributes to the development of many cancers. The tools for studying genome instability in mammalian cells are limited, whereas model organisms such as are more amenable to these studies. Here, we discuss the many genetic assays developed to measure the rate of occurrence of Gross Chromosomal Rearrangements (called GCRs) in These genetic assays have been used to identify many types of GCRs, including translocations, interstitial deletions, and broken chromosomes healed by telomere addition, and have identified genes that act in the suppression and formation of GCRs. Insights from these studies have contributed to the understanding of pathways and mechanisms that suppress genome instability and how these pathways cooperate with each other. Integrated models for the formation and suppression of GCRs are discussed.
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http://dx.doi.org/10.1534/genetics.112.145805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500125PMC
July 2017

Reconstitution of DNA polymerase ε-dependent mismatch repair with purified proteins.

Proc Natl Acad Sci U S A 2017 04 6;114(14):3607-3612. Epub 2017 Mar 6.

Ludwig Institute for Cancer Research, University of California School of Medicine, La Jolla, CA 92093-0669;

Mammalian and mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or the Msh2-MutS homolog 3 (Msh3) stimulates 5' to 3' excision by exonuclease 1 (Exo1) from a single-strand break 5' to the mispair, excising the mispair. In the other, Msh2-Msh6 or Msh2-Msh3 activate the MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3' to the mispair, to make nicks 5' to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase ε (Pol ε) may also play a role in MMR. Here we describe the reconstitution of Pol ε-dependent MMR using proteins. A mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze both short-patch and long-patch 5' nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3' to the mispair, a mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze an MMR reaction that required Mlh1-Pms1. These results demonstrate that Pol ε can act in eukaryotic MMR in vitro.
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http://dx.doi.org/10.1073/pnas.1701753114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389320PMC
April 2017

Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability.

Hum Mol Genet 2017 01;26(2):258-269

Department of Neurosciences, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA, USA.

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/β-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of β-tubulin to fold or become assembled into the α/β-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.
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http://dx.doi.org/10.1093/hmg/ddw383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075555PMC
January 2017

A genetic network that suppresses genome rearrangements in Saccharomyces cerevisiae and contains defects in cancers.

Nat Commun 2016 Apr 13;7:11256. Epub 2016 Apr 13.

Ludwig Institute for Cancer Res., University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0669, USA.

Gross chromosomal rearrangements (GCRs) play an important role in human diseases, including cancer. The identity of all Genome Instability Suppressing (GIS) genes is not currently known. Here multiple Saccharomyces cerevisiae GCR assays and query mutations were crossed into arrays of mutants to identify progeny with increased GCR rates. One hundred eighty two GIS genes were identified that suppressed GCR formation. Another 438 cooperatively acting GIS genes were identified that were not GIS genes, but suppressed the increased genome instability caused by individual query mutations. Analysis of TCGA data using the human genes predicted to act in GIS pathways revealed that a minimum of 93% of ovarian and 66% of colorectal cancer cases had defects affecting one or more predicted GIS gene. These defects included loss-of-function mutations, copy-number changes associated with reduced expression, and silencing. In contrast, acute myeloid leukaemia cases did not appear to have defects affecting the predicted GIS genes.
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http://dx.doi.org/10.1038/ncomms11256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833866PMC
April 2016

A personal historical view of DNA mismatch repair with an emphasis on eukaryotic DNA mismatch repair.

DNA Repair (Amst) 2016 Feb 3;38:3-13. Epub 2015 Dec 3.

Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, Moores-UCSD Cancer Center and Institute for Molecular Medicine, University of CA, San Diego School of Medicine, La Jolla, CA 92093-0669, United States. Electronic address:

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http://dx.doi.org/10.1016/j.dnarep.2015.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740188PMC
February 2016

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System.

J Biol Chem 2015 Aug 13;290(35):21580-90. Epub 2015 Jul 13.

From the Ludwig Institute for Cancer Research, the Department of Cellular and Molecular Medicine, Moores-UCSD Cancer Center, and the Institute of Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, California 92093

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.
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http://dx.doi.org/10.1074/jbc.M115.662189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571882PMC
August 2015

Exonuclease 1-dependent and independent mismatch repair.

DNA Repair (Amst) 2015 Aug 30;32:24-32. Epub 2015 Apr 30.

Ludwig Institute for Cancer Research, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA; Departments of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA; Moores - UCSD Cancer Center, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA; Institute of Genomic Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA. Electronic address:

DNA mismatch repair (MMR) acts to repair mispaired bases resulting from misincorporation errors during DNA replication and also recognizes mispaired bases in recombination (HR) intermediates. Exonuclease 1 (Exo1) is a 5' → 3' exonuclease that participates in a number of DNA repair pathways. Exo1 was identified as an exonuclease that participates in Saccharomyces cerevisiae and human MMR where it functions to excise the daughter strand after mispair recognition, and additionally Exo1 functions in end resection during HR. However, Exo1 is not absolutely required for end resection during HR in vivo. Similarly, while Exo1 is required in MMR reactions that have been reconstituted in vitro, genetics studies have shown that it is not absolutely required for MMR in vivo suggesting the existence of Exo1-independent and Exo1-dependent MMR subpathways. Here, we review what is known about the Exo1-independent and Exo1-dependent subpathways, including studies of mutations in MMR genes that specifically disrupt either subpathway.
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http://dx.doi.org/10.1016/j.dnarep.2015.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522362PMC
August 2015

New insights into the mechanism of DNA mismatch repair.

Chromosoma 2015 Dec 11;124(4):443-62. Epub 2015 Apr 11.

German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.

The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by up-to-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR.
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http://dx.doi.org/10.1007/s00412-015-0514-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600670PMC
December 2015

Loss of the thioredoxin reductase Trr1 suppresses the genomic instability of peroxiredoxin tsa1 mutants.

PLoS One 2014 23;9(9):e108123. Epub 2014 Sep 23.

Centre National de la Recherche Scientifique, UMR3348, Orsay, France; Institut Curie, Centre de Recherche, Orsay, France.

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a Can(R) mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the Can(R) mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108123PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172583PMC
December 2015

PCNA and Msh2-Msh6 activate an Mlh1-Pms1 endonuclease pathway required for Exo1-independent mismatch repair.

Mol Cell 2014 Jul 26;55(2):291-304. Epub 2014 Jun 26.

Ludwig Institute for Cancer Research, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Moores-UCSD Cancer Center, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Institute of Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA. Electronic address:

Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1)-dependent and -independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyperaccumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.
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http://dx.doi.org/10.1016/j.molcel.2014.04.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113420PMC
July 2014

Mlh2 is an accessory factor for DNA mismatch repair in Saccharomyces cerevisiae.

PLoS Genet 2014 May 8;10(5):e1004327. Epub 2014 May 8.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Cellular and Molecular Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Moores-UCSD Cancer Center, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.
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http://dx.doi.org/10.1371/journal.pgen.1004327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014439PMC
May 2014

S-score: a scoring system for the identification and prioritization of predicted cancer genes.

PLoS One 2014 7;9(4):e94147. Epub 2014 Apr 7.

Brain Institute, UFRN, Natal, Brazil.

A new method, which allows for the identification and prioritization of predicted cancer genes for future analysis, is presented. This method generates a gene-specific score called the "S-Score" by incorporating data from different types of analysis including mutation screening, methylation status, copy-number variation and expression profiling. The method was applied to the data from The Cancer Genome Atlas and allowed the identification of known and potentially new oncogenes and tumor suppressors associated with different clinical features including shortest term of survival in ovarian cancer patients and hormonal subtypes in breast cancer patients. Furthermore, for the first time a genome-wide search for genes that behave as oncogenes and tumor suppressors in different tumor types was performed. We envisage that the S-score can be used as a standard method for the identification and prioritization of cancer genes for follow-up studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094147PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978018PMC
December 2014
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