Publications by authors named "Fabio Puddu"

12 Publications

  • Page 1 of 1

Trajectory and uniqueness of mutational signatures in yeast mutators.

Proc Natl Acad Sci U S A 2020 10 23;117(40):24947-24956. Epub 2020 Sep 23.

Institut Curie, Paris Sciences et Lettres Research University, CNRS, UMR3244, 75248 Paris Cedex 05, France;

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the and backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2011332117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547211PMC
October 2020

Genome architecture and stability in the Saccharomyces cerevisiae knockout collection.

Nature 2019 09 11;573(7774):416-420. Epub 2019 Sep 11.

The Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, UK.

Despite major progress in defining the functional roles of genes, a complete understanding of their influences is far from being realized, even in relatively simple organisms. A major milestone in this direction arose via the completion of the yeast Saccharomyces cerevisiae gene-knockout collection (YKOC), which has enabled high-throughput reverse genetics, phenotypic screenings and analyses of synthetic-genetic interactions. Ensuing experimental work has also highlighted some inconsistencies and mistakes in the YKOC, or genome instability events that rebalance the effects of specific knockouts, but a complete overview of these is lacking. The identification and analysis of genes that are required for maintaining genomic stability have traditionally relied on reporter assays and on the study of deletions of individual genes, but whole-genome-sequencing technologies now enable-in principle-the direct observation of genome instability globally and at scale. To exploit this opportunity, we sequenced the whole genomes of nearly all of the 4,732 strains comprising the homozygous diploid YKOC. Here, by extracting information on copy-number variation of tandem and interspersed repetitive DNA elements, we describe-for almost every single non-essential gene-the genomic alterations that are induced by its loss. Analysis of this dataset reveals genes that affect the maintenance of various genomic elements, highlights cross-talks between nuclear and mitochondrial genome stability, and shows how strains have genetically adapted to life in the absence of individual non-essential genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1549-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774800PMC
September 2019

Phosphorylation of Histone H4T80 Triggers DNA Damage Checkpoint Recovery.

Mol Cell 2018 11 25;72(4):625-635.e4. Epub 2018 Oct 25.

The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK. Electronic address:

In response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2018.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242705PMC
November 2018

Detection of functional protein domains by unbiased genome-wide forward genetic screening.

Sci Rep 2018 04 18;8(1):6161. Epub 2018 Apr 18.

The Wellcome/CRUK Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, CB2 1QN, Cambridge, UK.

Establishing genetic and chemo-genetic interactions has played key roles in elucidating mechanisms by which certain chemicals perturb cellular functions. In contrast to gene disruption/depletion strategies to identify mechanisms of drug resistance, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations has been limited. Here, by demonstrating its utility in identifying chemical-genetic suppressors of sensitivity to the DNA topoisomerase I poison camptothecin or the poly(ADP-ribose) polymerase inhibitor olaparib, we detail an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast or haploid mammalian cells in a short timeframe, and with potential applications in other haploid systems. In addition to applications in molecular biology research, this protocol can be used to identify drug targets and predict drug-resistance mechanisms. Mapping suppressor mutations on the primary or tertiary structures of protein suppressor hits provides insights into functionally relevant protein domains. Importantly, we show that olaparib resistance is linked to missense mutations in the DNA binding regions of PARP1, but not in its catalytic domain. This provides experimental support to the concept of PARP1 trapping on DNA as the prime source of toxicity to PARP inhibitors, and points to a novel olaparib resistance mechanism with potential therapeutic implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-24400-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906580PMC
April 2018

Chromatin determinants impart camptothecin sensitivity.

EMBO Rep 2017 06 7;18(6):1000-1012. Epub 2017 Apr 7.

The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK

Camptothecin-induced locking of topoisomerase 1 on DNA generates a physical barrier to replication fork progression and creates topological stress. By allowing replisome rotation, absence of the Tof1/Csm3 complex promotes the conversion of impending topological stress to DNA catenation and causes camptothecin hypersensitivity. Through synthetic viability screening, we discovered that histone H4 K16 deacetylation drives the sensitivity of yeast cells to camptothecin and that inactivation of this pathway by mutating H4 K16 or the genes suppresses much of the hypersensitivity of strains towards this agent. We show that disruption of rDNA or telomeric silencing does not mediate camptothecin resistance but that disruption of Sir1-dependent chromatin domains is sufficient to suppress camptothecin sensitivity in wild-type and cells. We suggest that topoisomerase 1 inhibition in proximity of these domains causes topological stress that leads to DNA hypercatenation, especially in the absence of the Tof1/Csm3 complex. Finally, we provide evidence of the evolutionarily conservation of this mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201643560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452016PMC
June 2017

Synthetic viability genomic screening defines Sae2 function in DNA repair.

EMBO J 2015 Jun 21;34(11):1509-22. Epub 2015 Apr 21.

The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK The Wellcome Trust Sanger Institute, Hinxton Cambridge, UK

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201590973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474527PMC
June 2015

RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks.

Nat Struct Mol Biol 2011 Dec 4;19(1):17-24. Epub 2011 Dec 4.

Genome Stability Unit, Clare Hall Laboratories, London Research Institute, South Mimms, Hertfordshire, UK.

In higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ɛ onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.2177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306020PMC
December 2011

Sensing of replication stress and Mec1 activation act through two independent pathways involving the 9-1-1 complex and DNA polymerase ε.

PLoS Genet 2011 Mar 17;7(3):e1002022. Epub 2011 Mar 17.

Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, Milano, Italy.

Following DNA damage or replication stress, budding yeast cells activate the Rad53 checkpoint kinase, promoting genome stability in these challenging conditions. The DNA damage and replication checkpoint pathways are partially overlapping, sharing several factors, but are also differentiated at various levels. The upstream kinase Mec1 is required to activate both signaling cascades together with the 9-1-1 PCNA-like complex and the Dpb11 (hTopBP1) protein. After DNA damage, Dpb11 is also needed to recruit the adaptor protein Rad9 (h53BP1). Here we analyzed the mechanisms leading to Mec1 activation in vivo after DNA damage and replication stress. We found that a ddc1Δdpb11-1 double mutant strain displays a synthetic defect in Rad53 and H2A phosphorylation and is extremely sensitive to hydroxyurea (HU), indicating that Dpb11 and the 9-1-1 complex independently promote Mec1 activation. A similar phenotype is observed when both the 9-1-1 complex and the Dpb4 non-essential subunit of DNA polymerase ε (Polε) are contemporarily absent, indicating that checkpoint activation in response to replication stress is achieved through two independent pathways, requiring the 9-1-1 complex and Polε.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1002022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060063PMC
March 2011

Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation.

Mol Cell 2010 Oct;40(1):50-62

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, 20133, Italy.

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2010.09.004DOI Listing
October 2010

Dynamics of Rad9 chromatin binding and checkpoint function are mediated by its dimerization and are cell cycle-regulated by CDK1 activity.

PLoS Genet 2010 Aug 5;6(8). Epub 2010 Aug 5.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy.

Saccharomyces cerevisiae Rad9 is required for an effective DNA damage response throughout the cell cycle. Assembly of Rad9 on chromatin after DNA damage is promoted by histone modifications that create docking sites for Rad9 recruitment, allowing checkpoint activation. Rad53 phosphorylation is also dependent upon BRCT-directed Rad9 oligomerization; however, the crosstalk between these molecular determinants and their functional significance are poorly understood. Here we report that, in the G1 and M phases of the cell cycle, both constitutive and DNA damage-dependent Rad9 chromatin association require its BRCT domains. In G1 cells, GST or FKBP dimerization motifs can substitute to the BRCT domains for Rad9 chromatin binding and checkpoint function. Conversely, forced Rad9 dimerization in M phase fails to promote its recruitment onto DNA, although it supports Rad9 checkpoint function. In fact, a parallel pathway, independent on histone modifications and governed by CDK1 activity, allows checkpoint activation in the absence of Rad9 chromatin binding. CDK1-dependent phosphorylation of Rad9 on Ser11 leads to specific interaction with Dpb11, allowing Rad53 activation and bypassing the requirement for the histone branch.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1001047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916856PMC
August 2010

Checkpoint mechanisms at the intersection between DNA damage and repair.

DNA Repair (Amst) 2009 Sep 3;8(9):1055-67. Epub 2009 Jun 3.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.

In response to genomic insults cells trigger a signal transduction pathway, known as DNA damage checkpoint, whose role is to help the cell to cope with the damage by coordinating cell cycle progression, DNA replication and DNA repair mechanisms. Accumulating evidence suggests that activation of the first checkpoint kinase in the cascade is not due to the lesion itself, but it requires recognition and initial processing of the lesion by a specific repair mechanism. Repair enzymes likely convert a variety of physically and chemically different lesions to a unique common structure, a ssDNA region, which is the checkpoint triggering signal. Checkpoint kinases can modify the activity of repair mechanisms, allowing for efficient repair, on one side, and modulating the generation of the ssDNA signal, on the other. This strategy may be important to allow the most effective repair and a prompt recovery from the damage condition. Interestingly, at least in some cases, if the damage level is low enough the cell can deal with the lesions and it does not need to activate the checkpoint response. On the other hand if damage level is high or if the lesions are not rapidly repairable, checkpoint mechanisms become important for cell survival and preservation of genome integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2009.04.022DOI Listing
September 2009

Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint.

Mol Cell Biol 2008 Aug 9;28(15):4782-93. Epub 2008 Jun 9.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G(1) phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Delta dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00330-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493362PMC
August 2008