Abstract:Summary
Replication stress is a crucial driver of genomic instability. Understanding the mechanisms of replication stress response is instrumental to improve diagnosis and treatment of human disease. Electron microscopy (EM) is currently the only technique that allows to directly visualize a high number of replication intermediates and to monitor their remodeling upon stress. At the same time, DNA fiber analysis is useful to gain mechanistic insight on how genotoxic agents perturb replication fork dynamics gen… Show more
“…Whether or not transient accumulation and/or partial resection of reversed forks is visible by EM upon short genotoxic treatments in BRCA2 -defective cells likely reflects different kinetics of fork reversal and processing in different cell lines 18 . These data highlight the different resolution and limitations of DNA fiber assays and EM visualization of fork remodeling and degradation, as recently discussed 19 . Fig.…”
Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.
“…Whether or not transient accumulation and/or partial resection of reversed forks is visible by EM upon short genotoxic treatments in BRCA2 -defective cells likely reflects different kinetics of fork reversal and processing in different cell lines 18 . These data highlight the different resolution and limitations of DNA fiber assays and EM visualization of fork remodeling and degradation, as recently discussed 19 . Fig.…”
Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.
“…It is seemingly counterintuitive that inhibiting MRE11 activity rescues fork speed but leads to further accumulation of reversed forks upon inactivation of various DDR factors. However, it is important to note that DNA fiber spreading measures the rate of fork progression over a distance, while EM analysis provides snapshots of the most persisting intermediates along the path of active forks (Vindigni and Lopes, 2017). As discussed above, MRE11-dependent degradation of transiently reversed forks may counteract DNA synthesis under conditions of defective DDR, leading to slow fork progression as a net effect.…”
Section: Reduced Fork Speed Upon Rnf168 Depletion Depends On Nucleolymentioning
Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, ÎłH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.
“…In addition, lamins dysfunction could hinder the proper recruitment of replication fork protective factors upon fork stalling, causing replication stress-induced genomic instability ( Figure 3B ). To understand in-depth how lamins dysfunction affects DNA replication requires the utilization of newly developed techniques such as single-molecule replication assays (DNA fiber assays), iPOND ( I solation of P roteins O n N ascent D NA) [ 112 ], and electron microscopy [ 113 ], as well as depletion/reconstitution experiments to find strategies that rescue replication defects.…”
Section: Lamins As Caretakers Of the Genomementioning
Mammalian nuclei are equipped with a framework of intermediate filaments that function as a karyoskeleton. This nuclear scaffold, formed primarily by lamins (A-type and B-type), maintains the spatial and functional organization of the genome and of sub-nuclear compartments. Over the past decade, a body of evidence has highlighted the significance of these structural nuclear proteins in the maintenance of nuclear architecture and mechanical stability, as well as genome function and integrity. The importance of these structures is now unquestioned given the wide range of degenerative diseases that stem from LMNA gene mutations, including muscular dystrophy disorders, peripheral neuropathies, lipodystrophies, and premature aging syndromes. Here, we review our knowledge about how alterations in nuclear lamins, either by mutation or reduced expression, impact cellular mechanisms that maintain genome integrity. Despite the fact that DNA replication is the major source of DNA damage and genomic instability in dividing cells, how alterations in lamins function impact replication remains minimally explored. We summarize recent studies showing that lamins play a role in DNA replication, and that the DNA damage that accumulates upon lamins dysfunction is elicited in part by deprotection of replication forks. We also discuss the emerging model that DNA damage and replication stress are “sensed” at the cytoplasm by proteins that normally survey this space in search of foreign nucleic acids. In turn, these cytosolic sensors activate innate immune responses, which are materializing as important players in aging and cancer, as well as in the response to cancer immunotherapy.
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