ABSTRACT
Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress may also be applied to DNA-induced replication stalling.
Subject(s)
DNA Replication , DNA , DNA/genetics , DNA/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , Genomic Instability , Humans , Trinucleotide Repeats/geneticsABSTRACT
The breast cancer type-1 susceptibility protein (BRCA1) contributes to genome integrity through homologous recombinational DNA repair and by protecting stalled replication forks from nucleolytic degradation. We recently discovered that fork protection requires a conformational change of BRCA1 unimportant to homologous recombination repair, indicating separate roles for BRCA1 in these pathways.
ABSTRACT
The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.
Subject(s)
BRCA1 Protein/chemistry , BRCA1 Protein/metabolism , DNA Replication , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , BRCA1 Protein/genetics , Cell Line, Tumor , DNA Replication/genetics , Genomic Instability/genetics , Humans , Isomerism , Mutation , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , Phosphorylation , Phosphoserine/metabolism , Protein Binding , Rad51 Recombinase/metabolismABSTRACT
SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 focus retention and increases NHEJ and radioresistance. Collectively, our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.
Subject(s)
Cysteine Endopeptidases/metabolism , DNA End-Joining Repair/genetics , DNA Repair/genetics , Homologous Recombination/genetics , Sumoylation/genetics , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins , Cell Line, Tumor , Cell Survival/radiation effects , Cysteine Endopeptidases/genetics , DNA Breaks, Double-Stranded , HEK293 Cells , HeLa Cells , Humans , Infrared Rays , Nuclear Proteins/metabolism , Radiation Tolerance/genetics , Signal Transduction/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Valosin Containing Protein/metabolismABSTRACT
The opposing activities of 53BP1 and BRCA1 influence pathway choice in DNA double-strand-break repair. How BRCA1 counteracts the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2â¼ubiquitin and demonstrate that BRCA1-BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitination by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1-deficient cells. BRCA1-BARD1's function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin and optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning, and the need for SMARCAD1 in olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus, BRCA1-BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair.
Subject(s)
BRCA1 Protein/genetics , Chromatin/metabolism , DNA Helicases/genetics , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Recombinational DNA Repair , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , BRCA1 Protein/metabolism , Binding Sites , Camptothecin/pharmacology , Chromatin/chemistry , Chromatin/drug effects , Cloning, Molecular , DNA Breaks, Double-Stranded , DNA Cleavage/drug effects , DNA Helicases/metabolism , DNA, Neoplasm/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HeLa Cells , Histones/genetics , Histones/metabolism , Humans , Models, Molecular , Phthalazines/pharmacology , Piperazines/pharmacology , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effectsABSTRACT
Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.
