Publications by authors named "Fabio Vanoli"

17 Publications

  • Page 1 of 1

Interhomolog Homologous Recombination in Mouse Embryonic Stem Cells.

Methods Mol Biol 2021 ;2153:127-143

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Homologous recombination is a critical mechanism for the repair of DNA double-strand breaks (DSBs). It occurs predominantly between identical sister chromatids and at lower frequency can also occur between homologs. Interhomolog homologous recombination (IH-HR) has the potential lead to substantial loss of genetic information, i.e., loss of heterozygosity (LOH), when it is accompanied by crossing over. In this chapter, we describe a system to study IH-HR induced by a defined DSB in mouse embryonic stem cells derived from F1 hybrid mice. This system is based on the placement of mutant selectable marker genes, one of which contains an I-SceI endonuclease cleavage site, on the two homologs such that repair of the I-SceI-generated DSB from the homolog leads to drug resistance. Loss of heterozygosity arising during IH-HR is analyzed using a PCR-based approach. Finally, we present a strategy to analyze the role of BLM helicase in this system.
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http://dx.doi.org/10.1007/978-1-0716-0644-5_10DOI Listing
March 2021

Generation of chromosomal translocations that lead to conditional fusion protein expression using CRISPR-Cas9 and homology-directed repair.

Methods 2017 05 15;121-122:138-145. Epub 2017 May 15.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. Electronic address:

Recurrent chromosomal translocations often lead to expression of fusion proteins associated with oncogenic transformation. To study translocations and downstream events, genome editing techniques have been developed to generate chromosomal translocations through non-homologous end joining of DNA double-strand breaks introduced at the two participating endogenous loci. However, the frequencies at which these events occur is usually too low to efficiently clone cells carrying the translocation. This article provides a detailed method using CRISPR-Cas9 technology and homology-directed repair to efficiently isolate cells harboring a chromosomal translocation. For an additional level of control, the resulting fusion protein is conditionally expressed to allow early events in oncogenic transformation to be studied. We focus on the generation of the EWSR1-WT1 fusion using human mesenchymal cells, which is associated with the translocation found in desmoplastic small round cell tumors.
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http://dx.doi.org/10.1016/j.ymeth.2017.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531069PMC
May 2017

CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells.

Proc Natl Acad Sci U S A 2017 04 21;114(14):3696-3701. Epub 2017 Mar 21.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.
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http://dx.doi.org/10.1073/pnas.1700622114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389291PMC
April 2017

Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells.

Toxicol Sci 2016 09 22;153(1):70-8. Epub 2016 Jul 22.

*Wise Laboratory of Environmental and Genetic Toxicology, Portland, Maine 04104 Graduate School of Biomedical Science and Engineering, University of Maine, Orono, Maine 04469

Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis.
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http://dx.doi.org/10.1093/toxsci/kfw103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601504PMC
September 2016

Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks.

Nucleic Acids Res 2016 06 21;44(11):5204-17. Epub 2016 Mar 21.

Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam, 1105 AZ, The Netherlands Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing.
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http://dx.doi.org/10.1093/nar/gkw179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914091PMC
June 2016

Genomic Complexity Profiling Reveals That HORMAD1 Overexpression Contributes to Homologous Recombination Deficiency in Triple-Negative Breast Cancers.

Cancer Discov 2015 May 13;5(5):488-505. Epub 2015 Mar 13.

Breakthrough Breast Cancer Research Unit, King's College London, London, United Kingdom. Department of Research Oncology, King's Health Partners AHSC, Life Sciences and Medicine, King's College London, London, United Kingdom. The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom.

Unlabelled: Triple-negative breast cancers (TNBC) are characterized by a wide spectrum of genomic alterations, some of which might be caused by defects in DNA repair processes such as homologous recombination (HR). Despite this understanding, associating particular patterns of genomic instability with response to therapy has been challenging. Here, we show that allelic-imbalanced copy-number aberrations (AiCNA) are more prevalent in TNBCs that respond to platinum-based chemotherapy, thus providing a candidate predictive biomarker for this disease. Furthermore, we show that a high level of AiCNA is linked with elevated expression of a meiosis-associated gene, HORMAD1. Elevated HORMAD1 expression suppresses RAD51-dependent HR and drives the use of alternative forms of DNA repair, the generation of AiCNAs, as well as sensitizing cancer cells to HR-targeting therapies. Our data therefore provide a mechanistic association between HORMAD1 expression, a specific pattern of genomic instability, and an association with response to platinum-based chemotherapy in TNBC.

Significance: Previous studies have shown correlation between mutational "scars" and sensitivity to platinums extending beyond associations with BRCA1/2 mutation, but do not elucidate the mechanism. Here, a novel allele-specific copy-number characterization of genome instability identifies and functionally validates the inappropriate expression of the meiotic gene HORMAD1 as a driver of HR deficiency in TNBC, acting to induce allelic imbalance and moderate platinum and PARP inhibitor sensitivity with implications for the use of such "scars" and expression of meiotic genes as predictive biomarkers.
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http://dx.doi.org/10.1158/2159-8290.CD-14-1092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490184PMC
May 2015

Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.

Mol Cell 2015 Mar 5;57(5):812-823. Epub 2015 Feb 5.

IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy. Electronic address:

Chromosomal replication is entwined with DNA damage tolerance (DDT) and chromatin structure establishment via elusive mechanisms. Here we examined how specific replication conditions affecting replisome architecture and repriming impact on DDT. We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion. The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing. Notably, the DDT defects of Polα/Primase/Ctf4 mutants are not the consequence of increased sister chromatid distance, but are instead caused by altered single-stranded DNA metabolism and abnormal replication fork topology. We propose that error-free TS is driven by timely replicative helicase-coupled re-priming. Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.
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http://dx.doi.org/10.1016/j.molcel.2014.12.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352764PMC
March 2015

Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system.

Methods 2014 Sep 12;69(2):171-178. Epub 2014 Jun 12.

Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 USA.

Gene targeting - homologous recombination between transfected DNA and a chromosomal locus - is greatly stimulated by a DNA break in the target locus. Recently, the RNA-guided Cas9 endonuclease, involved in bacterial adaptive immunity, has been modified to function in mammalian cells. Unlike other site-specific endonucleases whose specificity resides within a protein, the specificity of Cas9-mediated DNA cleavage is determined by a guide RNA (gRNA) containing an ∼20 nucleotide locus-specific RNA sequence, representing a major advance for versatile site-specific cleavage of the genome without protein engineering. This article provides a detailed method using the Cas9 system to target expressed genes in mouse embryonic stem cells. In this method, a promoterless marker flanked by short homology arms to the target locus is transfected into cells together with Cas9 and gRNA expression vectors. Importantly, biallelic gene knockout is obtained at high frequency by only one round of targeting using a single marker.
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http://dx.doi.org/10.1016/j.ymeth.2014.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405113PMC
September 2014

Homologous recombination DNA repair genes play a critical role in reprogramming to a pluripotent state.

Cell Rep 2013 Mar 7;3(3):651-60. Epub 2013 Mar 7.

Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

Induced pluripotent stem cells (iPSCs) hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs). Additional mechanistic studies uncover a direct role of the homologous recombination (HR) pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.
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http://dx.doi.org/10.1016/j.celrep.2013.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315363PMC
March 2013

Noncanonical role of the 9-1-1 clamp in the error-free DNA damage tolerance pathway.

Mol Cell 2013 Feb 20;49(3):536-46. Epub 2012 Dec 20.

Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

Damaged DNA is an obstacle during DNA replication and a cause of genome instability and cancer. To bypass this problem, eukaryotes activate DNA damage tolerance (DDT) pathways that involve ubiquitylation of the DNA polymerase clamp proliferating cell nuclear antigen (PCNA). Monoubiquitylation of PCNA mediates an error-prone pathway by recruiting translesion polymerases, whereas polyubiquitylation activates an error-free pathway that utilizes undamaged sister chromatids as templates. The error-free pathway involves recombination-related mechanisms; however, the factors that act along with polyubiquitylated PCNA remain largely unknown. Here we report that the PCNA-related 9-1-1 complex, which is typically linked to checkpoint signaling, participates together with Exo1 nuclease in error-free DDT. Notably, 9-1-1 promotes template switching in a manner that is distinct from its canonical checkpoint functions and uncoupled from the replication fork. Our findings thus reveal unexpected cooperation in the error-free pathway between the two related clamps and indicate that 9-1-1 plays a broader role in the DNA damage response than previously assumed.
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http://dx.doi.org/10.1016/j.molcel.2012.11.016DOI Listing
February 2013

53BP1 is a haploinsufficient tumor suppressor and protects cells from radiation response in glioma.

Cancer Res 2012 Oct 21;72(20):5250-60. Epub 2012 Aug 21.

Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

The DNA damage response (DDR) plays a crucial role in tumor development in different tissues. Here, we show that p53-binding protein 1 (53BP1), a key element of the DDR, is heterozygously lost in approximately 20% of human glioblastoma multiforme (GBM) specimens, primarily of the Proneural subtype, and low 53BP1 expression levels are associated with worse prognosis. We present evidence that 53BP1 behaves as haploinsufficient tumor suppressor in a mouse model of platelet-derived growth factor-induced gliomagenesis. We also show that very low level of 53BP1 as found in 53BP1 null gliomas or robust 53BP1 gene silencing in glioma cell lines (but not 53BP1 heterozygous tumors or partial gene knockdown) sensitizes glioma cells to ionizing radiation (IR), both in vitro and in vivo. We further show the 53BP1 gene silencing induces defects in the nonhomologous end-joining (NHEJ) DNA repair pathway. These deficiencies lead to a failure to fully repair the damaged DNA upon exposure of glioma cells to IR with a consequent prolonged cell-cycle arrest and increased apoptosis. Our data suggest that either 53BP1 or other NHEJ components may be critical molecules to be pharmacologically targeted in GBM in combination with standard therapies.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-0045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771704PMC
October 2012

Molecular pathways: old drugs define new pathways: non-histone acetylation at the crossroads of the DNA damage response and autophagy.

Clin Cancer Res 2012 May 18;18(9):2436-42. Epub 2012 Apr 18.

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.

Histone deacetylases (HDAC) modulate acetylation and the function of histone and non-histone proteins. HDAC inhibitors have been developed to block the aberrant action of HDACs in cancer, and several are in clinical use (vorinostat, romidepsin, and valproic acid). Detailed understanding of their action is lacking, however, and their clinical activity is limited in most cases. Recently, HDACs have been involved in the control of the DNA damage response (DDR) at several levels and in directly regulating the acetylation of a number of DDR proteins (including CtIP and Exo1). Mechanistically, acetylation leads to the degradation of double-strand break repair enzymes through autophagy, providing a novel, direct link between DDR and autophagy. These observations, obtained in yeast cells, should now be translated to mammalian model systems and cancer cells to reveal whether this acetylation link is maintained in mammals, and if and how it is deregulated in cancer. In addition to HDACs, DDR and autophagy have been addressed pharmacologically, suggesting that the acetylation link, if involved in cancer, can be exploited for the design of new anticancer treatments.
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http://dx.doi.org/10.1158/1078-0432.CCR-11-0767DOI Listing
May 2012

Acetylation: a novel link between double-strand break repair and autophagy.

Cancer Res 2012 Mar;72(6):1332-5

Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.

Histone deacetylase (HDAC) inhibitors are clinically relevant because they are used as anticancer drugs. Recent evidence sheds light on an intriguing connection among the DNA damage response (DDR), protein acetylation, and autophagy. HDAC inhibitors have been shown to counteract key steps in the cellular response to double-strand break formation by affecting checkpoint activation, homologous recombination-mediated repair of DNA lesions, and stability of crucial enzymes involved in resection of DNA ends. The degradation of the resection factors depends on autophagy, which plays a detrimental role when cells are in a hyperacetylated state and experience treatment with radiomimetic anticancer drugs. Future work will be required to further investigate the mechanisms underlying the link between acetylation, autophagy, and the DDR, as well as the significance of mTORC1 inhibitors, which are potent inducers of autophagy that are now used in cancer treatment.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-3172DOI Listing
March 2012

HDACs link the DNA damage response, processing of double-strand breaks and autophagy.

Nature 2011 Mar;471(7336):74-79

Fondazione IFOM (Istituto FIRC di Oncologia Molecolare), IFOM-IEO Campus, via Adamello 16, Milan 20139, Italy.

Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA-RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA-RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.
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http://dx.doi.org/10.1038/nature09803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3935290PMC
March 2011

Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch.

PLoS Genet 2010 Nov 11;6(11):e1001205. Epub 2010 Nov 11.

Fondazione IFOM, Istituto FIRC di Oncologia Molecolare, Milan, Italy.

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.
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http://dx.doi.org/10.1371/journal.pgen.1001205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978687PMC
November 2010

SUMOylation regulates Rad18-mediated template switch.

Nature 2008 Dec;456(7224):915-20

IFOM, the FIRC Institute for Molecular Oncology Foundation, IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy.

Replication by template switch is thought to mediate DNA damage-bypass and fillings of gaps. Gap-filling repair requires homologous recombination as well as Rad18- and Rad5-mediated proliferating cell nuclear antigen (PCNA) polyubiquitylation. However, it is unclear whether these processes are coordinated, and the physical evidence for Rad18-Rad5-dependent template switch at replication forks is still elusive. Here we show, using genetic and physical approaches, that in budding yeast (Saccharomyces cerevisiae) Rad18 is required for the formation of X-shaped sister chromatid junctions (SCJs) at damaged replication forks through a process involving PCNA polyubiquitylation and the ubiquitin-conjugating enzymes Mms2 and Ubc13. The Rad18-Mms2-mediated damage-bypass through SCJs requires the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 and SUMOylated PCNA, and is coordinated with Rad51-dependent recombination events. We propose that the Rad18-Rad5-Mms2-dependent SCJs represent template switch events. Altogether, our results unmask a role for PCNA ubiquitylation and SUMOylation pathways in promoting transient damage-induced replication-coupled recombination events involving sister chromatids at replication forks.
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http://dx.doi.org/10.1038/nature07587DOI Listing
December 2008

Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing.

Oncogene 2004 Feb;23(6):1206-13

Istituto FIRC di Oncologia Molecolare, Via Adamello 16, Milano 20141, Italy.

The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.
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http://dx.doi.org/10.1038/sj.onc.1207199DOI Listing
February 2004