Homologous recombination (HR) is required for faithful repair of double strand breaks (DSBs) and is believed to be important for DNA replication under stressful conditions in unicellular organisms. ...However, its role during DNA replication in high eukaryotes has always been elusive. In particular, due to the essential nature of its main players it has been difficult to dissect the direct role of HR in DNA replication.
Recent studies revealed that some key HR factors such as Rad51 and BRCA2 play unexpected functions during DNA replication by protecting nascent DNA from Mre11 mediated degradation, which takes place at stalled replication forks. These novel functions appear to be essential to ensure smooth progression of DNA replication and to promote maintenance of genome stability.
Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication ...of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.
The development of metastatic cancer is a multistage process, which often requires decades to complete. Impairments in DNA damage control and DNA repair in cancer cell precursors generate genetically ...heterogeneous cell populations. However, despite heterogeneity most solid cancers have stereotypical behaviours, including invasiveness and suppression of immune responses that can be unleashed with immunotherapy targeting lymphocyte checkpoints. The mechanisms leading to the acquisition of stereotypical properties remain poorly understood. Reactivation of embryonic development processes in cells with unstable genomes might contribute to tumour expansion and metastasis formation. However, it is unclear whether these events are linked to immune response modulation. Tumours and embryos have non-self-components and need to avoid immune responses in their microenvironment. In mammalian embryos, neo-antigens are of paternal origin, while in tumour cells DNA mismatch repair and replication defects generate them. Inactivation of the maternal immune response towards the embryo, which occurs at the placental–maternal interface, is key to ensuring embryonic development. This regulation is accomplished by the trophoblast, which mimics several malignant cell features, including the ability to invade normal tissues and to avoid host immune responses, often adopting the same cancer immunoediting strategies. A better understanding as to whether and how genotoxic stress promotes cancer development through reactivation of programmes occurring during early stages of mammalian placentation could help to clarify resistance to drugs targeting immune checkpoint and DNA damage responses and to develop new therapeutic strategies to eradicate cancer.
To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a ...critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells.
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•SNF2 fork remodelers promote nascent DNA degradation in BRCA1/2-deficient cells•Fork remodelers favor MRE11 binding to stalled forks in BRCA1-deficient cells•Forks regressed by fork remodelers are degraded by MRE11 in BRCA1-deficient cells•Depletion of fork remodelers reduces genomic instability in BRCA1/2-deficient cells
Taglialatela et al. report that the replication fork remodelers SMARCAL1, ZRANB3, and HLTF promote the degradation of nascent DNA catalyzed by the MRE11 nuclease in BRCA1/2-deficient mammary epithelial cells treated with replication stress-inducing agents. Depletion of fork remodelers restores fork integrity and reduces genome instability in BRCA1/2-deficient cells.
GPS data suggest that the NW South America corner forms a semi‐rigid block drifting NE‐ward along the regional dextral strike‐slip faults that bound an oceanic terrane accreted in Late Cretaceous ...times to W Ecuador and Colombia. However, the relevance of both strike‐slip versus thrust tectonics during Cenozoic times and their relation with oceanic terrane accretion are unclear. Here we report on the paleomagnetism of 31 mid‐upper Eocene to upper Miocene mainly volcanic sites from the Cordilleras Occidental and Real of southern Ecuador. Eleven sites show that the western Cordillera Occidental underwent a 24° ± 10° clockwise (CW) rotation with respect to South America after late Miocene times, while no rotation occurred further east. We relate the regional CW rotation to the emplacement of the Cordillera Occidental nappe onto the continental sediments of the Interandean Valley, blanketing the Cordillera's eastern margin. As rotation and continental sedimentation onset ages are similar, we interpret such tectonic depression as a narrow flexural basin formed ahead of the advancing nappe front. The 20°–30° CW Neogene rotation of the Cordillera Occidental is indistinguishable from the post‐Cretaceous rotation of the Coastal forearc oceanic blocks, implying that the whole W Ecuador Andean chain was detached and rotated over a mid‐crustal detachment during the last 10 Ma. Eocene‐Miocene paleomagnetic inclination values are systematically consistent with those expected for South America, thus excluding latitudinal terrane drift. We suggest that the Andes of Ecuador and Peru form the “Ecuadorian Orocline”, formed by opposing orogenic rotations around the Amazonian craton indenter.
Key Points
20°–30°CW post‐Late Miocene rotation of the Cordillera Occidental, no rotation of the Cordillera Real, southern Ecuador
The southern Cordillera Occidental emplaced as a rotational nappe after the late Miocene‐Pliocene over the flexural Interandean Valley basin
No paleomagnetic evidence for latitudinal terrane drift and orogen‐parallel strike‐slip activity since Mid‐Late Eocene (∼40 Ma)
During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, ...the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.
Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, ...BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double‐strand break (DSB) repair and is in part due to the prevention of MRE11‐mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease‐mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA‐damaging agents. Here, we examine the mechanistic aspects of HR‐mediated fork protection, addressing the crosstalk between HR and replication proteins.
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.
The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg ...extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading- and lagging-strand DNA synthesis. In contrast, ssDNA gaps behind forks, which are prevalent on damaged templates, result from Mre11-dependent degradation of newly synthesized DNA strands and are suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct roles for Rad51 at replication forks, demonstrating that Rad51 protects newly synthesized DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.
Cell division cycle protein 45 (Cdc45) is required for DNA synthesis during genome duplication, as a component of the Cdc45-MCM-GINS (CMG) helicase. Despite its essential biological function, its ...biochemical role in DNA replication has remained elusive. Here we report the 2.1-Å crystal structure of human Cdc45, which confirms its evolutionary link with the bacterial RecJ nuclease and reveals several unexpected features that underpin its function in eukaryotic DNA replication. These include a long-range interaction between N- and C-terminal DHH domains, blocking access to the DNA-binding groove of its RecJ-like fold, and a helical insertion in its N-terminal DHH domain, which appears poised for replisome interactions. In combination with available electron microscopy data, we validate by mutational analysis the mechanism of Cdc45 association with the MCM ring and GINS co-activator, critical for CMG assembly. These findings provide an indispensable molecular basis to rationalize the essential role of Cdc45 in genomic duplication.
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