Publications by authors named "Christopher D Putnam"

61 Publications

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

Nat Commun 2021 09 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8458276PMC
September 2021

Strand discrimination in DNA mismatch repair.

DNA Repair (Amst) 2021 09 19;105:103161. Epub 2021 Jun 19.

San Diego Branch, Ludwig Institute for Cancer Research, La Jolla CA, 92093, United States; Department of Medicine, University of California School of Medicine, La Jolla CA, 92093, United States. Electronic address:

DNA mismatch repair (MMR) corrects non-Watson-Crick basepairs generated by replication errors, recombination intermediates, and some forms of chemical damage to DNA. In MutS and MutL homolog-dependent MMR, damaged bases do not identify the error-containing daughter strand that must be excised and resynthesized. In organisms like Escherichia coli that use methyl-directed MMR, transient undermethylation identifies the daughter strand. For other organisms, growing in vitro and in vivo evidence suggest that strand discrimination is mediated by DNA replication-associated daughter strand nicks that direct asymmetric loading of the replicative clamp (the β-clamp in bacteria and the proliferating cell nuclear antigen, PCNA, in eukaryotes). Structural modeling suggests that replicative clamps mediate strand specificity either through the ability of MutL homologs to recognize the fixed orientation of the daughter strand relative to one face of the replicative clamps or through parental strand-specific diffusion of replicative clamps on DNA, which places the daughter strand in the MutL homolog endonuclease active site. Finally, identification of bacteria that appear to lack strand discrimination mediated by a replicative clamp and a pre-existing nick suggest that other strand discrimination mechanisms exist or that these organisms perform MMR by generating a double-stranded DNA break intermediate, which may be analogous to NucS-mediated MMR.
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http://dx.doi.org/10.1016/j.dnarep.2021.103161DOI Listing
September 2021

A Case of Severe Combined Immunodeficiency Missed by Newborn Screening.

J Clin Immunol 2021 08 12;41(6):1352-1355. Epub 2021 Mar 12.

Department of Pediatrics, Division of Hematology/Oncology, Washington University School of Medicine, St. Louis, MO, USA.

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http://dx.doi.org/10.1007/s10875-021-01020-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954206PMC
August 2021

Immunodeficiency and bone marrow failure with mosaic and germline TLR8 gain of function.

Blood 2021 05;137(18):2450-2462

Division of Allergy and Immunology, Department of Pediatrics, University of Arkansas for Medical Sciences and Arkansas Children's Hospital, Little Rock, AR.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.
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http://dx.doi.org/10.1182/blood.2020009620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109013PMC
May 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

Mechanisms underlying genome instability mediated by formation of foldback inversions in .

Elife 2020 08 7;9. Epub 2020 Aug 7.

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

Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here, we used a genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by , , , and and large-loop hairpins that were suppressed by , , , and . Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, -dependent break-induced replication did not appear to be involved forming secondary rearrangements.
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http://dx.doi.org/10.7554/eLife.58223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467729PMC
August 2020

MutS sliding clamps on an uncertain track to DNA mismatch repair.

Proc Natl Acad Sci U S A 2020 08 4;117(34):20351-20353. Epub 2020 Aug 4.

San Diego Branch, Ludwig Institute for Cancer Research, La Jolla, CA 92093;

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http://dx.doi.org/10.1073/pnas.2013560117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456105PMC
August 2020

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

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

Mutations in topoisomerase IIβ result in a B cell immunodeficiency.

Nat Commun 2019 08 13;10(1):3644. Epub 2019 Aug 13.

Department of Pediatrics, University of California at San Diego, La Jolla, CA, 92093, USA.

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization.
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http://dx.doi.org/10.1038/s41467-019-11570-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692411PMC
August 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

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

EPCAM mutation update: Variants associated with congenital tufting enteropathy and Lynch syndrome.

Hum Mutat 2019 02 29;40(2):142-161. Epub 2018 Nov 29.

Department of Pediatrics, University of California, San Diego, La Jolla, California.

The epithelial cell adhesion molecule gene (EPCAM, previously known as TACSTD1 or TROP1) encodes a membrane-bound protein that is localized to the basolateral membrane of epithelial cells and is overexpressed in some tumors. Biallelic mutations in EPCAM cause congenital tufting enteropathy (CTE), which is a rare chronic diarrheal disorder presenting in infancy. Monoallelic deletions of the 3' end of EPCAM that silence the downstream gene, MSH2, cause a form of Lynch syndrome, which is a cancer predisposition syndrome associated with loss of DNA mismatch repair. Here, we report 13 novel EPCAM mutations from 17 CTE patients from two separate centers, review EPCAM mutations associated with CTE and Lynch syndrome, and structurally model pathogenic missense mutations. Statistical analyses indicate that the c.499dupC (previously reported as c.498insC) frameshift mutation was associated with more severe treatment regimens and greater mortality in CTE, whereas the c.556-14A>G and c.491+1G>A splice site mutations were not correlated with treatments or outcomes significantly different than random simulation. These findings suggest that genotype-phenotype correlations may be useful in contributing to management decisions of CTE patients. Depending on the type and nature of EPCAM mutation, one of two unrelated diseases may occur, CTE or Lynch syndrome.
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http://dx.doi.org/10.1002/humu.23688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328345PMC
February 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

Guinier peak analysis for visual and automated inspection of small-angle X-ray scattering data.

J Appl Crystallogr 2016 Oct 4;49(Pt 5):1412-1419. Epub 2016 Aug 4.

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

The Guinier region in small-angle X-ray scattering (SAXS) defines the radius of gyration, , and the forward scattering intensity, (0). In Guinier peak analysis (GPA), the plot of () transforms the Guinier region into a characteristic peak for visual and automated inspection of data. Deviations of the peak position from the theoretical position in dimensionless GPA plots can suggest parameter errors, problematic low-resolution data, some kinds of intermolecular interactions or elongated scatters. To facilitate automated analysis by GPA, the elongation ratio (ER), which is the ratio of the areas in the pair-distribution function () after and before the () maximum, was characterized; symmetric samples have ER values around 1, and samples with ER values greater than 5 tend to be outliers in GPA analysis. Use of GPA+ER can be a helpful addition to SAXS data analysis pipelines.
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http://dx.doi.org/10.1107/S1600576716010906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045725PMC
October 2016

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

Evolution of the methyl directed mismatch repair system in Escherichia coli.

DNA Repair (Amst) 2016 Feb 2;38:32-41. Epub 2015 Dec 2.

Ludwig Institute for Cancer Research and University of California San Diego School of Medicine, 9500 Gilman Drive, La Jolla, San Diego, CA 92093-0669, United States. Electronic address:

DNA mismatch repair (MMR) repairs mispaired bases in DNA generated by replication errors. MutS or MutS homologs recognize mispairs and coordinate with MutL or MutL homologs to direct excision of the newly synthesized DNA strand. In most organisms, the signal that discriminates between the newly synthesized and template DNA strands has not been definitively identified. In contrast, Escherichia coli and some related gammaproteobacteria use a highly elaborated methyl-directed MMR system that recognizes Dam methyltransferase modification sites that are transiently unmethylated on the newly synthesized strand after DNA replication. Evolution of methyl-directed MMR is characterized by the acquisition of Dam and the MutH nuclease and by the loss of the MutL endonuclease activity. Methyl-directed MMR is present in a subset of Gammaproteobacteria belonging to the orders Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales, and a subset of the Alteromonadales (the EPVAA group) as well as in gammaproteobacteria that have obtained these genes by horizontal gene transfer, including the medically relevant bacteria Fluoribacter, Legionella, and Tatlockia and the marine bacteria Methylophaga and Nitrosococcus.
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http://dx.doi.org/10.1016/j.dnarep.2015.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740232PMC
February 2016

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

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

DNA repair pathway selection caused by defects in TEL1, SAE2, and de novo telomere addition generates specific chromosomal rearrangement signatures.

PLoS Genet 2014 Apr 3;10(4):e1004277. Epub 2014 Apr 3.

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

Whole genome sequencing of cancer genomes has revealed a diversity of recurrent gross chromosomal rearrangements (GCRs) that are likely signatures of specific defects in DNA damage response pathways. However, inferring the underlying defects has been difficult due to insufficient information relating defects in DNA metabolism to GCR signatures. By analyzing over 95 mutant strains of Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants. The hph-retaining GCRs isolated from tel1Δ mutants contained either an interstitial deletion dependent on non-homologous end-joining or an inverted duplication that appeared to be initiated from a double strand break (DSB) on chrV L followed by hairpin formation, copying of chrV L from the DSB toward the centromere, and homologous recombination to capture the hph-containing end of chrV L. In contrast, hph-containing GCRs from other mutants were primarily interstitial deletions (mrc1Δ tof1Δ) or inverted duplications (sae2Δ and rad53Δ sml1Δ). Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs. Both types of hph-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of hph retention were due to the relative efficiencies of competing DNA repair pathways. Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.
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http://dx.doi.org/10.1371/journal.pgen.1004277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3974649PMC
April 2014

A saccharomyces cerevisiae RNase H2 interaction network functions to suppress genome instability.

Mol Cell Biol 2014 Apr 18;34(8):1521-34. Epub 2014 Feb 18.

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

Errors during DNA replication are one likely cause of gross chromosomal rearrangements (GCRs). Here, we analyze the role of RNase H2, which functions to process Okazaki fragments, degrade transcription intermediates, and repair misincorporated ribonucleotides, in preventing genome instability. The results demonstrate that rnh203 mutations result in a weak mutator phenotype and cause growth defects and synergistic increases in GCR rates when combined with mutations affecting other DNA metabolism pathways, including homologous recombination (HR), sister chromatid HR, resolution of branched HR intermediates, postreplication repair, sumoylation in response to DNA damage, and chromatin assembly. In some cases, a mutation in RAD51 or TOP1 suppressed the increased GCR rates and/or the growth defects of rnh203Δ double mutants. This analysis suggests that cells with RNase H2 defects have increased levels of DNA damage and depend on other pathways of DNA metabolism to overcome the deleterious effects of this DNA damage.
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http://dx.doi.org/10.1128/MCB.00960-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993591PMC
April 2014

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

PLoS Genet 2013 Oct 31;9(10):e1003869. Epub 2013 Oct 31.

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

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.
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http://dx.doi.org/10.1371/journal.pgen.1003869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814310PMC
October 2013
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