► Replication stress induces strand-annealing events called template switching. ► Fork stalling, DNA gaps and breaks can trigger template switching. ► Template switching can be either error-free or ...faulty in its outcome. ► Template switching can promote gap filling and fork restart. ► Ubiquitin-family modifications regulate damage-induced template switching.
Homologous recombination plays an important role in the maintenance of genome integrity. Arrested forks and DNA lesions trigger strand annealing events, called template switching, which can provide for accurate damage bypass, but can also lead to chromosome rearrangements. Advances have been made in understanding the underlying mechanisms for these events and in elucidating the factors involved. Ubiquitin- and SUMO-mediated modification pathways have emerged as key players in regulating damage-induced template switching. Here I review the biological significance of template switching at the nexus of DNA replication and recombination, and the role of ubiquitin-like modifications in mediating and controlling this process.
The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific ...DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.
Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One ...mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research.
The error‐free DNA damage tolerance (DDT) pathway is crucial for replication completion and genome integrity. Mechanistically, this process is driven by a switch of templates accompanied by sister ...chromatid junction (SCJ) formation. Here, we asked if DDT intermediate processing is temporarily regulated, and what impact such regulation may have on genome stability. We find that persistent DDT recombination intermediates are largely resolved before anaphase through a G2/M damage checkpoint‐independent, but Cdk1/Cdc5‐dependent pathway that proceeds via a previously described Mus81‐Mms4‐activating phosphorylation. The Sgs1‐Top3‐ and Mus81‐Mms4‐dependent resolution pathways occupy different temporal windows in relation to replication, with the Mus81‐Mms4 pathway being restricted to late G2/M. Premature activation of the Cdk1/Cdc5/Mus81 pathway, achieved here with phosphomimetic Mms4 variants as well as in S‐phase checkpoint‐deficient genetic backgrounds, induces crossover‐associated chromosome translocations and precocious processing of damage‐bypass SCJ intermediates. Taken together, our results underscore the importance of uncoupling error‐free versus erroneous recombination intermediate processing pathways during replication, and establish a new paradigm for the role of the DNA damage response in regulating genome integrity by controlling crossover timing.
The structure‐specific endonuclease Mus81‐Mms4, which resolves persistent replication intermediates prior to chromosome segregation, requires tight cell‐cycle restriction to not jeopardize genomic integrity during S phase.
The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered ...during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.
Abstract
Chronic low levels of survival motor neuron (SMN) protein cause spinal muscular atrophy (SMA). SMN is ubiquitously expressed, but the mechanisms underlying predominant neuron degeneration in ...SMA are poorly understood. We report that chronic low levels of SMN cause Senataxin (SETX)-deficiency, which results in increased RNA-DNA hybrids (R-loops) and DNA double-strand breaks (DSBs), and deficiency of DNA-activated protein kinase-catalytic subunit (DNA-PKcs), which impairs DSB repair. Consequently, DNA damage accumulates in patient cells, SMA mice neurons and patient spinal cord tissues. In dividing cells, DSBs are repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) pathways, but neurons predominantly use NHEJ, which relies on DNA-PKcs activity. In SMA dividing cells, HR repairs DSBs and supports cellular proliferation. In SMA neurons, DNA-PKcs-deficiency causes defects in NHEJ-mediated repair leading to DNA damage accumulation and neurodegeneration. Restoration of SMN levels rescues SETX and DNA-PKcs deficiencies and DSB accumulation in SMA neurons and patient cells. Moreover, SETX overexpression in SMA neurons reduces R-loops and DNA damage, and rescues neurodegeneration. Our findings identify combined deficiency of SETX and DNA-PKcs stemming downstream of SMN as an underlying cause of DSBs accumulation, genomic instability and neurodegeneration in SMA and suggest SETX as a potential therapeutic target for SMA.
encodes an iron-sulfur cluster DNA helicase required for development, mutated, and overexpressed in cancers. Here, we show that loss of
causes replication stress and sensitizes cancer cells to DNA ...damaging agents, including poly ADP ribose polymerase (PARP) inhibitors and platinum drugs. We find that DDX11 helicase activity prevents chemotherapy drug hypersensitivity and accumulation of DNA damage. Mechanistically, DDX11 acts downstream of 53BP1 to mediate homology-directed repair and RAD51 focus formation in manners nonredundant with BRCA1 and BRCA2. As a result,
down-regulation aggravates the chemotherapeutic sensitivity of
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-mutated cancers and resensitizes chemotherapy drug-resistant
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-mutated cancer cells that regained homologous recombination proficiency. The results further indicate that DDX11 facilitates recombination repair by assisting double strand break resection and the loading of both RPA and RAD51 on single-stranded DNA substrates. We propose DDX11 as a potential target in cancers by creating pharmacologically exploitable DNA repair vulnerabilities.
Similar to ubiquitin, SUMO forms chains, but the identity of SUMO-chain-modified factors and the purpose of this modification remain largely unknown. Here, we identify the budding yeast SUMO protease ...Ulp2, able to disassemble SUMO chains, as a DDK interactor enriched at replication origins that promotes DNA replication initiation. Replication-engaged DDK is SUMOylated on chromatin, becoming a degradation-prone substrate when Ulp2 no longer protects it against SUMO chain assembly. Specifically, SUMO chains channel DDK for SUMO-targeted ubiquitin ligase Slx5/Slx8-mediated and Cdc48 segregase-assisted proteasomal degradation. Importantly, the SUMOylation-defective ddk-KR mutant rescues inefficient replication onset and MCM activation in cells lacking Ulp2, suggesting that SUMO chains time DDK degradation. Using two unbiased proteomic approaches, we further identify subunits of the MCM helicase and other factors as SUMO-chain-modified degradation-prone substrates of Ulp2 and Slx5/Slx8. We thus propose SUMO-chain/Ulp2-protease-regulated proteasomal degradation as a mechanism that times the availability of functionally engaged SUMO-modified protein pools during replication and beyond.
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•Chromatin-bound DDK engaged in replication is SUMOylated at multiple sites•SUMO chains target DDK for STUbL-mediated Cdc48-assisted proteasomal degradation•SUMO protease Ulp2 protects DDK from degradation, allowing early steps of replication•Ulp2 curbs SUMO chain assembly, thus timing protein turnover in replication and beyond
Psakhye et al. uncover that SUMO chains specifically counteracted by the Ulp2 SUMO protease function like a countdown timer when they are assembled on substrates of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL), which channels them for proteasomal degradation, as demonstrated here for SUMOylated DDK engaged in DNA replication initiation.
Structural maintenance of chromosomes (SMCs) complexes, cohesin, condensin, and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, ...chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability of PDS5 null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality of PDS5-deleted cells can also be bypassed by simultaneous loss of the proliferating cell nuclear antigen (PCNA) unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO chain assembly, which we propose to time the availability of SMC complexes on chromatin.
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•SUMO chains target SMC complexes and are antagonized by the SUMO protease Ulp2•Essential role of the cohesin regulatory subunit Pds5 is to counteract SUMO chains•Fusion of Ulp2 to cohesin subunit kleisin Scc1 supports viability in the absence of Pds5•Ulp2 curbs SUMO chain assembly, thus timing the turnover of the SMC complexes
How the chromatin abundance of SMC complexes is regulated remains poorly understood. Psakhye and Branzei identify SUMO protease Ulp2 as a guardian of cohesin, condensin, and Smc5/6 complexes against SUMO-chain-targeted turnover. The authors show that the essential role of Pds5 cohesin subunit is to antagonize SUMO chains jointly with Ulp2.
A new study by Longo, Roy et al. has solved the structure of the RAD51C‐XRCC3 (CX3) heterodimer with a bound ATP analog, identifying two main structural interfaces and defining separable replication ...fork stability roles. One function relates to the ability of RAD51C to bind and assemble CX3 on nascent DNA, with an impact on the ability of forks to restart upon replication stress. The other relates to effective CX3 heterodimer formation, required for 5′ RAD51 filament capping, with effects on RAD51 filament disassembly, fork protection and influencing the persistence of reversed forks.
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