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1.
Nucleic Acids Res ; 2024 Jun 29.
Article in English | MEDLINE | ID: mdl-38943334

ABSTRACT

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.

2.
J Vis Exp ; (180)2022 02 03.
Article in English | MEDLINE | ID: mdl-35188138

ABSTRACT

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.


Subject(s)
DNA Repair , DNA Replication , DNA/genetics , DNA, Single-Stranded , Humans , S Phase
3.
Mol Cell ; 81(19): 4026-4040.e8, 2021 10 07.
Article in English | MEDLINE | ID: mdl-34624216

ABSTRACT

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.


Subject(s)
DNA Breaks, Single-Stranded , DNA Primase/metabolism , DNA Repair , DNA Replication , DNA, Neoplasm/biosynthesis , DNA-Directed DNA Polymerase/metabolism , G2 Phase , Multifunctional Enzymes/metabolism , Neoplasms/metabolism , S Phase , Antineoplastic Agents/pharmacology , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , Cell Line, Tumor , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Primase/genetics , DNA, Neoplasm/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , Genomic Instability , HEK293 Cells , Humans , MRE11 Homologue Protein/genetics , MRE11 Homologue Protein/metabolism , Multifunctional Enzymes/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Time Factors , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
4.
Mol Cell ; 81(4): 649-658, 2021 02 18.
Article in English | MEDLINE | ID: mdl-33515486

ABSTRACT

Accurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming. Here, we discuss recent advances toward our understanding of the mechanisms that regulate the fine-tuned balance between these different replication stress response pathways. We also discuss the molecular pathways required to fill single-stranded DNA gaps that accumulate throughout the genome after repriming and the biological consequences of using repriming instead of other DNA damage tolerance pathways on genome integrity and cell fitness.


Subject(s)
DNA Breaks, Single-Stranded , DNA Repair , DNA Replication , DNA, Single-Stranded/metabolism , Genomic Instability , Animals , DNA, Single-Stranded/genetics , Humans
5.
Crit Rev Biochem Mol Biol ; 56(1): 17-30, 2021 02.
Article in English | MEDLINE | ID: mdl-33179522

ABSTRACT

DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome. The Primase and DNA-directed Polymerase (PRIMPOL) is a recently discovered enzyme that possesses both primase and polymerase activities. PRIMPOL is emerging as a key player in DNA damage tolerance, particularly in vertebrate and human cells. Here, we review our current understanding of the function of PRIMPOL in DNA damage tolerance by focusing on the structural aspects that define its dual enzymatic activity, as well as on the mechanisms that control its chromatin recruitment and expression levels. We also focus on the latest findings on the mitochondrial and nuclear functions of PRIMPOL and on the impact of loss of these functions on genome stability and cell survival. Defining the function of PRIMPOL in DNA damage tolerance is becoming increasingly important in the context of human disease. In particular, we discuss recent evidence pointing at the PRIMPOL pathway as a novel molecular target to improve cancer cell response to DNA-damaging chemotherapy and as a predictive parameter to stratify patients in personalized cancer therapy.


Subject(s)
DNA Damage/genetics , DNA Primase/genetics , DNA Primase/metabolism , DNA Replication/genetics , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , Cell Nucleus/metabolism , Cell Survival/genetics , Chromatin/metabolism , DNA/genetics , DNA/metabolism , DNA Primase/chemistry , DNA-Directed DNA Polymerase/chemistry , Gene Knockdown Techniques , Genomic Instability , Humans , Mitochondria/metabolism , Multifunctional Enzymes/chemistry
6.
Mol Cell ; 77(3): 461-474.e9, 2020 02 06.
Article in English | MEDLINE | ID: mdl-31676232

ABSTRACT

Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.


Subject(s)
DNA Primase/metabolism , DNA Replication/drug effects , DNA-Directed DNA Polymerase/metabolism , Multifunctional Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line, Tumor , DNA/genetics , DNA Damage/genetics , DNA Damage/physiology , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Primase/physiology , DNA Replication/genetics , DNA Replication/physiology , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/physiology , HEK293 Cells , Humans , Multifunctional Enzymes/physiology , Ubiquitin-Protein Ligases/genetics
7.
Nucleic Acids Res ; 47(3): 1294-1310, 2019 02 20.
Article in English | MEDLINE | ID: mdl-29917110

ABSTRACT

Pds5 is required for sister chromatid cohesion, and somewhat paradoxically, to remove cohesin from chromosomes. We found that Pds5 plays a critical role during DNA replication that is distinct from its previously known functions. Loss of Pds5 hinders replication fork progression in unperturbed human and mouse cells. Inhibition of MRE11 nuclease activity restores fork progression, suggesting that Pds5 protects forks from MRE11-activity. Loss of Pds5 also leads to double-strand breaks, which are again reduced by MRE11 inhibition. The replication function of Pds5 is independent of its previously reported interaction with BRCA2. Unlike Pds5, BRCA2 protects forks from nucleolytic degradation only in the presence of genotoxic stress. Moreover, our iPOND analysis shows that the loading of Pds5 and other cohesion factors on replication forks is not affected by the BRCA2 status. Pds5 role in DNA replication is shared by the other cohesin-removal factor Wapl, but not by the cohesin complex component Rad21. Interestingly, depletion of Rad21 in a Pds5-deficient background rescues the phenotype observed upon Pds5 depletion alone. These findings support a model where loss of either component of the cohesin releasin complex perturbs cohesin dynamics on replication forks, hindering fork progression and promoting MRE11-dependent fork slowing.


Subject(s)
DNA Replication/genetics , MRE11 Homologue Protein/genetics , Nuclear Proteins/genetics , Phosphoproteins/genetics , BRCA2 Protein/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Chromatids/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA Damage/genetics , DNA-Binding Proteins , Deoxyribonucleases/genetics , Humans , Sister Chromatid Exchange/genetics , Cohesins
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