PRIMPOL-Mediated Adaptive Response Suppresses Replication Fork Reversal in BRCA-Deficient Cells.

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.

Dynamic stem loop extension by Pol θ and templated insertion during DNA repair.

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 single-stranded DNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both single-stranded DNA and RNA substrates by unimolecular stem loop synthesis initiated by only two 3' 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 RPA. 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 amongst 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.

Perturbing cohesin dynamics drives MRE11 nuclease-dependent replication fork slowing.

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.

Regulating Polθ in Breast Cancer.

DNA polymerase θ, a protein encoded by the POLQ 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 POLQ expression and TMEJ activity: the transcription factor, ZEB1.See related article by Prodhomme et al., p. 1595.

When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer.

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.

Mitotic regulators TPX2 and Aurora A protect DNA forks during replication stress by counteracting 53BP1 function.

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.

DNA Fiber Analysis: Mind the Gap!

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.