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1.
Sci Adv ; 8(5): eabk0221, 2022 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-35119917

RESUMEN

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.


Asunto(s)
Síndrome de Bloom , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Humanos , Proteínas Nucleares/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Recombinación Genética , Quinasa Tipo Polo 1
2.
Mol Cell ; 77(3): 528-541.e8, 2020 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-31759821

RESUMEN

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.


Asunto(s)
Replicación del ADN/fisiología , Estructuras R-Loop/genética , Recombinasa Rad51/metabolismo , Línea Celular Tumoral , ADN Ligasas/metabolismo , ADN Polimerasa III/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Células HeLa , Humanos , Estructuras R-Loop/fisiología , Recombinasa Rad51/genética , Recombinasa Rad51/fisiología , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , RecQ Helicasas/metabolismo , RecQ Helicasas/fisiología , Transcripción Genética/genética
3.
Nat Commun ; 8(1): 1333, 2017 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-29109511

RESUMEN

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.


Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Metilación , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Dímeros de Pirimidina/metabolismo , ARN Interferente Pequeño/genética , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/prevención & control , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Rayos Ultravioleta/efectos adversos
4.
EMBO J ; 36(22): 3372-3386, 2017 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-29018037

RESUMEN

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.


Asunto(s)
Ensamble y Desensamble de Cromatina , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Factor de Transcripción TFIIH/metabolismo , Rayos Ultravioleta , Xerodermia Pigmentosa/metabolismo , Muerte Celular/efectos de la radiación , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/efectos de la radiación , Genoma Humano , Células HEK293 , Células HeLa , Humanos , Nucleosomas/efectos de la radiación , Dímeros de Pirimidina/metabolismo , ARN Interferente Pequeño/metabolismo
5.
Front Genet ; 7: 68, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27200078

RESUMEN

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.

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