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.
DNA Fiber Analysis: Mind the Gap Quinet, Annabel; Carvajal-Maldonado, Denisse; Lemacon, Delphine ...
Methods in enzymology,
2017, Letnik:
591
Journal Article
Recenzirano
Understanding the mechanisms of replication stress response following genotoxic stress induction is rapidly emerging as a central theme in cell survival and human disease. The DNA fiber assay is one ...of the most powerful tools to study alterations in replication fork dynamics genome-wide at single-molecule resolution. This approach relies on the ability of many organisms to incorporate thymidine analogs into replicating DNA and is widely used to study how genotoxic agents perturb DNA replication. Here, we review different approaches available to prepare DNA fibers and discuss important limitations of each approach. We also review how DNA fiber analysis can be used to shed light upon several replication parameters including fork progression, restart, termination, and new origin firing. Next, we discuss a modified DNA fiber protocol to monitor the presence of single-stranded DNA (ssDNA) gaps on ongoing replication forks. ssDNA gaps are very common intermediates of several replication stress response mechanisms, but they cannot be detected by standard DNA fiber approaches due to the resolution limits of this technique. We discuss a novel strategy that relies on the use of an ssDNA-specific endonuclease to nick the ssDNA gaps and generate shorter DNA fibers that can be used as readout for the presence of ssDNA gaps. Finally, we describe a follow-up DNA fiber approach that can be used to study how ssDNA gaps are repaired postreplicatively.
DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and ...recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3'-5' exonuclease, 5' nuclease, or end-trimming nuclease) or lyase (5' dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.
Regulating Polθ in Breast Cancer Carvajal-Maldonado, Denisse; Wood, Richard D
Cancer research,
2021-Mar-15, 2021-03-15, Letnik:
81, Številka:
6
Journal Article
Recenzirano
Odprti dostop
DNA polymerase θ, a protein encoded by the
gene, is the defining factor for the DNA double-strand break repair pathway known as theta-mediated end-joining (TMEJ). Some cancers depend on TMEJ for ...survival and tumor growth. TMEJ might be useful as a biomarker to guide patient treatment and is now an active target for drug development, making it critical to understand how it is regulated in cells. In a recent article, Prodhomme and colleagues provide the first identification of a transcription regulator of
expression and TMEJ activity: the transcription factor, ZEB1.
.
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.
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•Multiple cisplatin doses suppress reversed fork degradation in BRCA-deficient cells•The PRIMPOL adaptive response suppresses fork reversal and leads to ssDNA gaps•The ATR kinase regulates the PRIMPOL-mediated adaptive response•Impaired fork reversal shifts the balance toward PRIMPOL-mediated repriming
Reversed replication forks are degraded in BRCA-deficient cells. Quinet et al. show that treatment with multiple cisplatin doses promotes PRIMPOL repriming while suppressing fork reversal and preventing degradation. These studies identify a new function of PRIMPOL in the replication stress response and explain how cells adapt to multiple drug doses.
Theta-mediated end joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend ssDNA ...oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both ssDNA and RNA substrates by unimolecular stem–loop synthesis initiated by only two 3ʹ terminal base pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem–loop synthesis competes with bimolecular end joining, even when a longer terminal microhomology for end joining is available. Both reactions are partially suppressed by the ssDNA-binding protein replication protein A. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem–loop synthesis. The ability to perform stem–loop synthesis from a minimally paired primer is rare among human DNA polymerases, but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem–loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.
53BP1 is a chromatin-associated protein that regulates the DNA damage response. In this study, we identify the TPX2/Aurora A heterodimer, nominally considered a mitotic kinase complex, as a novel ...binding partner of 53BP1. We find that TPX2/Aurora A plays a previously unrecognized role in DNA damage repair and replication fork stability by counteracting 53BP1 function. Loss of TPX2 or Aurora A compromises DNA end resection, BRCA1 and Rad51 recruitment, and homologous recombination. Furthermore, loss of TPX2 or Aurora A causes deprotection of stalled replication forks upon replication stress induction. This fork protection pathway counteracts MRE11 nuclease activity but functions in parallel to BRCA1. Strikingly, concurrent loss of 53BP1 rescues not only BRCA1/Rad51 recruitment but also the fork instability induced upon TPX2 loss. Our work suggests the presence of a feedback mechanism by which 53BP1 is regulated by a novel binding partner and uncovers a unique role for 53BP1 in replication fork stability.
Genomic instability caused by improper chromosomal segregation, aneuploidy and DNA damage is exceedingly amplified in tumor cells. Recently, replication stress has emerged as an important contributor ...to tumorigenesis. Most notably, cancer cells depend on replication stress response mechanisms to proliferate, thereby providing a strategy for their clearance. This work uncovers a previously unappreciated role of two known chromosomal segregation protein complexes during replication stress. The first is the releasin complex, formed by the Pds5 and Wapl proteins, which is required to unload cohesin from DNA prior to chromosomal separation. The second is the TPX2/Aurora-A complex, which promotes microtubule nucleation during mitosis. The results presented here show that the aberrant unloading of cohesin from replication forks associated with loss of either component of the releasin complex, Pds5 or Wapl, hinders replication fork progression and leads to extensive MRE11 nuclease-dependent degradation of the newly replicated strands. This defect leads to double-strand break accumulation and increased genomic instability. Loss of cohesin rescues the replication defect of Pds5 or Wapl-deficient cells, suggesting that Pds5 and Wapl are required to remove cohesin ahead of replication forks to ensure normal fork progression. These results revisit the functions of cohesin factors during replication and provide a new rationale for the recently identified tumor’s suppressor function of Pds5B. The results presented here also show that the TPX2/Aurora-A complex is recruited to chromatin by the DNA repair protein 53BP1. The complex promotes fork stability by preventing nascent strand resection by MRE11 under conditions of mild replication stress, similar to what has been previously reported for BRCA1. Loss of TPX2 in a BRCA1 null background potentiates the degradation phenotype, suggesting that TPX2 acts in a separate pathway from BRCA1 to promote replication fork protection. Strikingly, concurrent loss of TPX2 and 53BP1 rescues the fork instability phenotype induced by TPX2 loss, pointing to a novel role for 53BP1 during replication stress. Furthermore, loss of TPX2 in a BRCA1-deficient background also significantly decreases cell viability. Collectively, these results suggest that the TPX2/Aurora-A complex represents a novel attractive target to increase chemo-sensitivity in BRCA1-deficient tumors.
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