Biomedical subjects

Giordano Liberi

Publications and source records attributed to Giordano Liberi.

12 recordsLinked to original sources

PubMed2006

Ubc9- and mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks.

The Ubc9 SUMO-conjugating enzyme and the Siz1 SUMO ligase sumoylate several repair and recombination proteins, including PCNA. Sumoylated PCNA binds Srs2, a helicase counteracting certain recombination events. Here we show that ubc9 mutants depend on checkpoint, recombination, and replication genes for growth. ubc9 cells maintain stalled-fork stability but exhibit a Rad51-dependent accumulation of cruciform structures during replication of damaged templates. Mutations in the Mms21 SUMO ligase resemble the ubc9 mutations. However, siz1, srs2, or pcna mutants altered in sumoylation do not exhibit the ubc9/mms21 phenotype. Like ubc9/mms21 mutants, sgs1 and top3 mutants also accumulate X molecules at damaged forks, and Sgs1/BLM is sumoylated. We propose that Ubc9 and Mms21 act in concert with Sgs1 to resolve the X structures formed during replication. Our results indicate that Ubc9- and Mms21-mediated sumoylation functions as a regulatory mechanism, different from that of replication checkpoints, to prevent pathological accumulation of cruciform structures at damaged forks.

DNA Damage↗

PubMed2006

Methods to study replication fork collapse in budding yeast.

Replication of the eukaryotic genome is a difficult task, as cells must coordinate chromosome replication with chromatin remodeling, DNA recombination, DNA repair, transcription, cell cycle progression, and sister chromatid cohesion. Yet, DNA replication is a potentially genotoxic process, particularly when replication forks encounter a bulge in the template: forks under these conditions may stall and restart or even break down leading to fork collapse. It is now clear that fork collapse stimulates chromosomal rearrangements and therefore represents a potential source of DNA damage. Hence, the comprehension of the mechanisms that preserve replication fork integrity or that promote fork collapse are extremely relevant for the understanding of the cellular processes controlling genome stability. Here we describe some experimental approaches that can be used to physically visualize the quality of replication forks in the yeast S. cerevisiae and to distinguish between stalled and collapsed forks.

Blotting, Southern↗

PubMed2005

Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase.

S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by preventing unscheduled mitotic recombination events. We have studied the RecQ helicase-mediated mechanisms controlling genome stability by analyzing replication forks encountering a damaged template in sgs1 cells. We show that, in sgs1 mutants, recombination-dependent cruciform structures accumulate at damaged forks. Their accumulation requires Rad51 protein, is counteracted by Srs2 DNA helicase, and does not prevent fork movement. Sgs1, but not Srs2, promotes resolution of these recombination intermediates. A functional Rad53 checkpoint kinase that is known to protect the integrity of the sister chromatid junctions is required for the accumulation of recombination intermediates in sgs1 mutants. Finally, top3 and top3 sgs1 mutants accumulate the same structures as sgs1 cells. We suggest that, in sgs1 cells, the unscheduled accumulation of Rad51-dependent cruciform structures at damaged forks result from defective maturation of recombination-dependent intermediates that originate from the replication-related sister chromatid junctions. Our findings might contribute to explaining some of the recombination defects of BLM cells.

Adenosine Triphosphatases↗

PubMed2005

Srs2 and Sgs1 DNA helicases associate with Mre11 in different subcomplexes following checkpoint activation and CDK1-mediated Srs2 phosphorylation.

Mutations in the genes encoding the BLM and WRN RecQ DNA helicases and the MRE11-RAD50-NBS1 complex lead to genome instability and cancer predisposition syndromes. The Saccharomyces cerevisiae Sgs1 RecQ helicase and the Mre11 protein, together with the Srs2 DNA helicase, prevent chromosome rearrangements and are implicated in the DNA damage checkpoint response and in DNA recombination. By searching for Srs2 physical interactors, we have identified Sgs1 and Mre11. We show that Srs2, Sgs1, and Mre11 form a large complex, likely together with yet unidentified proteins. This complex reorganizes into Srs2-Mre11 and Sgs1-Mre11 subcomplexes following DNA damage-induced activation of the Mec1 and Tel1 checkpoint kinases. The defects in subcomplex formation observed in mec1 and tel1 cells can be recapitulated in srs2-7AV mutants that are hypersensitive to intra-S DNA damage and are altered in the DNA damage-induced and Cdk1-dependent phosphorylation of Srs2. Altogether our observations indicate that Mec1- and Tel1-dependent checkpoint pathways modulate the functional interactions between Srs2, Sgs1, and Mre11 and that the Srs2 DNA helicase represents an important target of the Cdk1-mediated cellular response induced by DNA damage.

Blotting, Western↗

PubMed2004

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1.

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

CDC2 Protein Kinase↗

PubMed2004

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

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.

Cell Cycle Proteins↗

PubMed2004

Initiation of DNA replication: a new hint from archaea.

In this issue, Robinson and coworkers provide new insights into the mechanisms of initiation of chromosome replication in Archea. This and other studies, focused on model organisms, will certainly help to understand how the replication process has evolved in Eukaryotes.

Archaea↗

PubMed2003

Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast.

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.

Crossing Over, Genetic↗

PubMed2003

Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms.

Cells overcome intra-S DNA damage and replication impediments by coupling chromosome replication to sister chromatid-mediated recombination and replication-bypass processes. Further, molecular junctions between replicated molecules have been suggested to assist sister chromatid cohesion until anaphase. Using two-dimensional gel electrophoresis, we have identified, in yeast cells, replication-dependent X-shaped molecules that appear during origin activation, branch migrate, and distribute along the replicon through a mechanism influenced by the rate of fork progression. Their formation is independent of Rad51- and Rad52-mediated homologous recombination events and is not affected by DNA damage or replication blocks. Further, in hydroxyurea-treated rad53 mutants, altered in the replication checkpoint, the branched molecules progressively degenerate and likely contribute to generate pathological structures. We suggest that cells couple sister chromatid tethering with replication initiation by generating specialized joint molecules resembling hemicatenanes: this process might prime cohesion and assist sister chromatid-mediated recombination and replication events.

Cell Cycle Proteins↗

PubMed2002

A dominant-negative MEC3 mutant uncovers new functions for the Rad17 complex and Tel1.

The Rad17-Mec3-Ddc1 complex is essential for the cellular response to genotoxic agents and is thought to be important for sensing DNA lesions. Deletion of any of the RAD17, MEC3 or DDC1 genes abolishes the G(1) and G(2) and impairs the intra-S DNA-damage checkpoints. We characterize a dominant-negative mec3-dn mutation that has an unexpected phenotype. It inactivates the G(1) checkpoint while it leaves the G(2) response functional, thus revealing a difference in the requirements of the DNA-damage response in different phases of the cell cycle. In an attempt to identify the molecular defect imparted by the mutation, we dissected step-by-step the signaling cascade, which is triggered by DNA lesions and requires the activity of Mec1 and Rad53 kinases. The analysis of the phosphorylation state of checkpoint factors and critical protein interactions showed that, in mec3-dn cells, the signal transduction cascade is triggered normally, and the central kinase Mec1 can be activated. In G(1) cells expressing the mutation, the signaling cannot proceed any further along the pathway, indicating that the Rad17 complex acts after the activation of Mec1, possibly recruiting targets for the kinase. We also show that the function of the G(2) checkpoint in mutant cells is maintained by an uncharacterized activity of Tel1, the yeast homologue of ATM. This work thus reports a previously undiscovered role for Tel1 in checkpoint control.

Cell Cycle↗

PubMed2002

Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase.

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.

Adaptation, Physiological↗

PubMedDate not supplied

Mechanisms controlling the integrity of replicating chromosomes in budding yeast.

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.

Cell Cycle↗