Biomedical subjects

Massimo Lopes

Publications and source records attributed to Massimo Lopes.

8 recordsLinked to original sources

PubMed2026

Cohesin reshapes replication fork contacts to aid fork slowing and reversal.

DNA replication forks can be challenged by cancer chemotherapeutic treatments, leading to accumulation of single-stranded DNA and slowdown of DNA synthesis. The marked plasticity of replication forks under replication stress ensures fork stability, damage tolerance and complete genome duplication1. Initiation and progression of replication forks occur in a three-dimensionally organized genome. DNA loop extrusion by the cohesin complex organizes the genome2 and regulates the initiation and positioning of DNA replication origins3,4. Although transient interaction of sister forks was recently reported during unperturbed replication5, the functional relevance of fork contacts during replication stress and the role of cohesin in this context remain unknown. Here we show that cohesin-mediated loop extrusion rearranges nascent DNA contacts at stressed replication forks to promote genome stability. Using auxin-inducible degron6, separation-of-function mutants7-9 and a newly developed Micro-C-based technique to capture chromatin contacts at nascent DNA (Repli-C), we found that loop-extruding cohesin accumulates at stalled replication forks, limiting sister-fork coupling in favour of inter-replicon contacts. This process promotes active fork slowing and reversal by preventing PRIMPOL action on single-stranded DNA1. These findings show that the replication stress response is not merely an accumulation of individual regulatory events, but is topologically integrated across the genome through cohesin loop extrusion. While providing a new function for loop-extruding cohesin, our results indicate the potential impact on cancer therapy of frequent cohesin mutations in tumours10.

Journal Article↗

PubMed2006

Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.

DNA replication forks pause in front of lesions on the template, eventually leading to cytotoxic chromosomal rearrangements. The in vivo structure of damaged eukaryotic replication intermediates has been so far elusive. Combining electron microscopy (EM) and two-dimensional (2D) gel electrophoresis, we found that UV-irradiated S. cerevisiae cells uncouple leading and lagging strand replication at irreparable UV lesions, thus generating long ssDNA regions on one side of the fork. Furthermore, small ssDNA gaps accumulate along replicated duplexes, likely resulting from repriming events downstream of the lesions on both leading and lagging strands. Translesion synthesis and homologous recombination counteract gap accumulation, without affecting fork progression. The DNA damage checkpoint contributes to gap repair and maintains a replication-competent fork structure. We propose that the coordinated action of checkpoint, recombination, and translesion synthesis-mediated processes at the fork and behind the fork preserves the integrity of replicating chromosomes by allowing efficient replication restart and filling the resulting ssDNA gaps.

Chromosomes↗

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

Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells.

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.

Cell Cycle↗

PubMed2004

Pol12, the B subunit of DNA polymerase alpha, functions in both telomere capping and length regulation.

The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, as a new factor involved in this process. Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing. Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1. Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping. These data reveal a novel function for Pol12 and a new connection between DNA polymerase alpha and Stn1. We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.

Cell Cycle Proteins↗

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

Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

Cell Cycle Proteins↗