RESUMO
DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme that removes RNA from RNA-DNA heteroduplexes. RTF2, like RNase H2, is essential for mammalian development and maintains normal replication speed. However, persistent RTF2 and RNase H2 at stalled replication forks prevent efficient replication restart, which is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for RTF2-dependent regulation of replication-coupled ribonucleotide removal and reveal the existence of PRIM1-mediated direct replication restart in mammalian cells.
Assuntos
Replicação do DNA , DNA , Animais , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteínas de Ciclo Celular/metabolismo , RNA/genética , Ribonucleases/metabolismo , Mamíferos/genéticaRESUMO
Genetic information is duplicated via the highly regulated process of DNA replication. The machinery coordinating this process, the replisome, encounters many challenges, including replication fork-stalling lesions that threaten the accurate and timely transmission of genetic information. Cells have multiple mechanisms to repair or bypass lesions that would otherwise compromise DNA replication1,2. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2) function to regulate Replication Termination Factor 2 (RTF2) at the stalled replisome, allowing for replication fork stabilization and restart3. Here we show that RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme responsible for removing RNA in the context of RNA-DNA heteroduplexes4-6. We show that during unperturbed DNA replication, RTF2, like RNase H2, is required to maintain normal replication fork speeds. However, persistent RTF2 and RNase H2 at stalled replication forks compromises the replication stress response, preventing efficient replication restart. Such restart is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for regulation of replication-coupled ribonucleotide incorporation during normal replication and the replication stress response that is achieved through RTF2. We also provide evidence for PRIM1 function in direct replication restart following replication stress in mammalian cells.
RESUMO
Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1-TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Microcefalia/genética , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Sítios de Ligação , Calorimetria , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Dano ao DNA , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Fibroblastos , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Mutação , Domínios e Motivos de Interação entre Proteínas , Serina Proteases/genética , Serina Proteases/metabolismo , Complexo Shelterina , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genéticaRESUMO
Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscore broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy.
Assuntos
Sistemas CRISPR-Cas/genética , Mutagênese/genética , Células-Tronco Pluripotentes/citologia , RNA/genética , Edição de Genes , Humanos , Mutação com Perda de Função/genética , RNA Guia de Cinetoplastídeos , TransfecçãoRESUMO
Telomeres use shelterin to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), repressing ataxia-telangiectasia, mutated (ATM) and ATM and Rad3-related (ATR) dependent DNA damage checkpoint responses. The MRE11 nuclease is thought to be essential for the resection of the 5' C-strand to generate the microhomologies necessary for alternative non-homologous end joining (A-NHEJ) repair. In the present study, we uncover DNA damage signaling and repair pathways engaged by components of the replisome complex to repair dysfunctional telomeres. In cells lacking MRN, single-stranded telomeric overhangs devoid of POT1-TPP1 do not recruit replication protein A (RPA), ATR-interacting protein (ATRIP), and RAD 51. Rather, components of the replisome complex, including Claspin, Proliferating cell nuclear antigen (PCNA), and Downstream neighbor of SON (DONSON), initiate DNA-PKcs-mediated p-CHK1 activation and A-NHEJ repair. In addition, Claspin directly interacts with TRF2 and recruits EXO1 to newly replicated telomeres to promote 5' end resection. Our data indicate that MRN is dispensable for the repair of dysfunctional telomeres lacking POT1-TPP1 and highlight the contributions of the replisome in telomere repair.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Telômero/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aminopeptidases/deficiência , Aminopeptidases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Quinase 1 do Ponto de Checagem/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/deficiência , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Exodesoxirribonucleases/metabolismo , Células HEK293 , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , Complexos Multienzimáticos/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Serina Proteases/deficiência , Serina Proteases/metabolismo , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
Telomeres are nucleoprotein complexes that play essential roles in protecting chromosome ends. Mammalian telomeres consist of repetitive DNA sequences bound by the shelterin complex. In this complex, the POT1-TPP1 heterodimer binds to single-stranded telomeric DNAs, while TRF1 and TRF2-RAP1 interact with double-stranded telomeric DNAs. TIN2, the linchpin of this complex, simultaneously interacts with TRF1, TRF2, and TPP1 to mediate the stable assembly of the shelterin complex. However, the molecular mechanism by which TIN2 interacts with these proteins to orchestrate telomere protection remains poorly understood. Here, we report the crystal structure of the N-terminal domain of TIN2 in complex with TIN2-binding motifs from TPP1 and TRF2, revealing how TIN2 interacts cooperatively with TPP1 and TRF2. Unexpectedly, TIN2 contains a telomeric repeat factor homology (TRFH)-like domain that functions as a protein-protein interaction platform. Structure-based mutagenesis analyses suggest that TIN2 plays an important role in maintaining the stable shelterin complex required for proper telomere end protection.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Serina Proteases/metabolismo , Complexo Shelterina/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/química , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Humanos , Camundongos , Conformação Proteica , Serina Proteases/química , Serina Proteases/isolamento & purificação , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/isolamento & purificação , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/isolamento & purificaçãoRESUMO
Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2TRFH domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.