ABSTRACT
Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G2-M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability.
Subject(s)
Bloom Syndrome , Cell Cycle Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Bloom Syndrome/genetics , Bloom Syndrome/metabolism , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Carrier Proteins/genetics , DNA-Binding Proteins/metabolism , Genomic Instability , Humans , Nuclear Proteins/metabolism , RecQ Helicases/genetics , RecQ Helicases/metabolism , Recombination, Genetic , Polo-Like Kinase 1ABSTRACT
Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.
Subject(s)
DNA Replication/physiology , R-Loop Structures/genetics , Rad51 Recombinase/metabolism , Cell Line, Tumor , DNA Ligases/metabolism , DNA Polymerase III/metabolism , DNA Replication/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases/genetics , Endonucleases/metabolism , HeLa Cells , Humans , R-Loop Structures/physiology , Rad51 Recombinase/genetics , Rad51 Recombinase/physiology , Rad52 DNA Repair and Recombination Protein/metabolism , RecQ Helicases/metabolism , RecQ Helicases/physiology , Transcription, Genetic/geneticsABSTRACT
Global-genome nucleotide excision repair (GG-NER) prevents ultraviolet (UV) light-induced skin cancer by removing mutagenic cyclobutane pyrimidine dimers (CPDs). These lesions are formed abundantly on DNA wrapped around histone octamers in nucleosomes, but a specialized damage sensor known as DDB2 ensures that they are accessed by the XPC initiator of GG-NER activity. We report that DDB2 promotes CPD excision by recruiting the histone methyltransferase ASH1L, which methylates lysine 4 of histone H3. In turn, methylated H3 facilitates the docking of the XPC complex to nucleosomal histone octamers. Consequently, DDB2, ASH1L and XPC proteins co-localize transiently on histone H3-methylated nucleosomes of UV-exposed cells. In the absence of ASH1L, the chromatin binding of XPC is impaired and its ability to recruit downstream GG-NER effectors diminished. Also, ASH1L depletion suppresses CPD excision and confers UV hypersensitivity. These findings show that ASH1L configures chromatin for the effective handoff between damage recognition factors during GG-NER activity.
Subject(s)
DNA Damage , DNA Repair , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Cell Line , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , HeLa Cells , Histone-Lysine N-Methyltransferase , Histones/metabolism , Humans , Methylation , Nucleosomes/metabolism , Protein Interaction Domains and Motifs , Pyrimidine Dimers/metabolism , RNA, Small Interfering/genetics , Skin Neoplasms/etiology , Skin Neoplasms/metabolism , Skin Neoplasms/prevention & control , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Ultraviolet Rays/adverse effectsABSTRACT
Ultraviolet (UV) light induces mutagenic cyclobutane pyrimidine dimers (CPDs) in nucleosomal DNA that is tightly wrapped around histone octamers. How global-genome nucleotide excision repair (GG-NER) processes CPDs despite that this chromatin arrangement is poorly understood. An increased chromatin association of CHD1 (chromodomain helicase DNA-binding 1) upon UV irradiation indicated possible roles of this chromatin remodeler in the UV damage response. Immunoprecipitation of chromatin fragments revealed that CHD1 co-localizes in part with GG-NER factors. Chromatin fractionation showed that the UV-dependent recruitment of CHD1 occurs to UV lesions in histone-assembled nucleosomal DNA and that this CHD1 relocation requires the lesion sensor XPC (xeroderma pigmentosum group C). In situ immunofluorescence analyses further demonstrate that CHD1 facilitates substrate handover from XPC to the downstream TFIIH (transcription factor IIH). Consequently, CHD1 depletion slows down CPD excision and sensitizes cells to UV-induced cytotoxicity. The finding of a CHD1-driven lesion handover between sequentially acting GG-NER factors on nucleosomal histone octamers suggests that chromatin provides a recognition scaffold enabling the detection of a subset of CPDs.
Subject(s)
Chromatin Assembly and Disassembly , DNA Damage , DNA Helicases/metabolism , DNA Repair/radiation effects , DNA-Binding Proteins/metabolism , Nucleosomes/metabolism , Transcription Factor TFIIH/metabolism , Ultraviolet Rays , Xeroderma Pigmentosum/metabolism , Cell Death/radiation effects , Chromatin/metabolism , Chromatin Assembly and Disassembly/radiation effects , Genome, Human , HEK293 Cells , HeLa Cells , Humans , Nucleosomes/radiation effects , Pyrimidine Dimers/metabolism , RNA, Small Interfering/metabolismABSTRACT
Global-genome nucleotide excision repair (GG-NER) prevents genome instability by excising a wide range of different DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV) light or intracellular side products of metabolism. As a versatile damage sensor, xeroderma pigmentosum group C (XPC) protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4(DDB2) and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4(DDB2) or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.
