The function of BCL11B in base excision repair contributes to its dual role as an oncogene and a haplo-insufficient tumor suppressor gene.

Genetic studies in mice and human cancers established BCL11B as a haploinsufficient tumor suppressor gene. Paradoxically, BCL11B is overexpressed in some human cancers where its knockdown is synthetic lethal. We identified the BCL11B protein in a proximity-dependent biotinylation screen performed with the DNA glycosylase NTHL1. In vitro DNA repair assays demonstrated that both BCL11B and a small recombinant BCL11B213-560 protein lacking transcription regulation potential can stimulate the enzymatic activities of two base excision repair (BER) enzymes: NTHL1 and Pol ß. In cells, BCL11B is rapidly recruited to sites of DNA damage caused by laser microirradiation. BCL11B knockdown delays, whereas ectopic expression of BCL11B213-560 accelerates, the repair of oxidative DNA damage. Inactivation of one BCL11B allele in TK6 lymphoblastoid cells causes an increase in spontaneous and radiation-induced mutation rates. In turn, ectopic expression of BCL11B213-560 cooperates with the RAS oncogene in cell transformation by reducing DNA damage and cellular senescence. These findings indicate that BCL11B functions as a BER accessory factor, safeguarding normal cells from acquiring mutations. Paradoxically, it also enables the survival of cancer cells that would otherwise undergo senescence or apoptosis due to oxidative DNA damage resulting from the elevated production of reactive oxygen species.

Differential effects of pesticides on dioxin receptor signaling and p53 activation.

As modern agricultural practices increase their use of chemical pesticides, it is inevitable that we will find a number of these xenobiotics within drinking water supplies and disseminated throughout the food chain. A major problem that arises from this pollution is that the effects of most of these pesticides on cellular mechanisms in general, and how they interact with each other and affect human cells are still poorly understood. In this study we make use of cultured human cancer cells to measure by qRT-PCR how pesticides affect gene expression of stress pathways. Immunoblotting studies were performed to monitor protein expression levels and activation of signaling pathways. We make use of immunofluorescence and microscopy to visualize and quantify DNA damage events in those cells. In the current study, we evaluate the potential of a subset of widely used pesticides to activate the dioxin receptor pathway and affect its crosstalk with estrogen receptor signaling. We quantify the impact of these chemicals on the p53-dependent cellular stress response. We find that, not only can the different pesticides activate the dioxin receptor pathway, most of them have better than additive effects on this pathway when combined at low doses. We also show that different pesticides have the ability to trigger crosstalk events that may generate genotoxic estrogen metabolites. Finally, we show that some, but not all of the tested pesticides can induce a p53-dependent stress response. Taken together our results provide evidence that several xenobiotics found within the environment have the potential to interact together to elicit significant effects on cell systems. Our data warrants caution when the toxicity of substances that are assessed simply for individual chemicals, since important biological effects could be observed only in the presence of other compounds, and that even at very low concentrations.

Chronic treatment with ATR and CHK1 inhibitors does not substantially increase the mutational burden of human cells.

DNA replication stress (RS) entails the frequent slow down and arrest of replication forks by a variety of conditions that hinder accurate and processive genome duplication. Elevated RS leads to genome instability, replication catastrophe and eventually cell death. RS is particularly prevalent in cancer cells and its exacerbation to unsustainable levels by chemotherapeutic agents remains a cornerstone of cancer treatments. The adverse consequences of RS are normally prevented by the ATR and CHK1 checkpoint kinases that stabilize stressed forks, suppress origin firing and promote cell cycle arrest when replication is perturbed. Specific inhibitors of these kinases have been developed and shown to potentiate RS and cell death in multiple in vitro cancer settings. Ongoing clinical trials are now probing their efficacy against various cancer types, either as single agents or in combination with mainstay chemotherapeutics. Despite their promise as valuable additions to the anti-cancer pharmacopoeia, we still lack a genome-wide view of the potential mutagenicity of these new drugs. To investigate this question, we performed chronic long-term treatments of TP53-depleted human cancer cells with ATR and CHK1 inhibitors (ATRi, AZD6738/ceralasertib and CHK1i, MK8776/SCH-900776). ATR or CHK1 inhibition did not significantly increase the mutational burden of cells, nor generate specific mutational signatures. Indeed, no notable changes in the numbers of base substitutions, short insertions/deletions and larger scale rearrangements were observed despite induction of replication-associated DNA breaks during treatments. Interestingly, ATR inhibition did induce a slight increase in closely-spaced mutations, a feature previously attributed to translesion synthesis DNA polymerases. The results suggest that ATRi and CHK1i do not have substantial mutagenic effects in vitro when used as standalone agents.

The PRP19 Ubiquitin Ligase, Standing at the Cross-Roads of mRNA Processing and Genome Stability.

mRNA processing factors are increasingly being recognized as important regulators of genome stability. By preventing and resolving RNA:DNA hybrids that form co-transcriptionally, these proteins help avoid replication-transcription conflicts and thus contribute to genome stability through their normal function in RNA maturation. Some of these factors also have direct roles in the activation of the DNA damage response and in DNA repair. One of the most intriguing cases is that of PRP19, an evolutionarily conserved essential E3 ubiquitin ligase that promotes mRNA splicing, but also participates directly in ATR activation, double-strand break resection and mitosis. Here, we review historical and recent work on PRP19 and its associated proteins, highlighting their multifarious cellular functions as central regulators of spliceosome activity, R-loop homeostasis, DNA damage signaling and repair and cell division. Finally, we discuss open questions that are bound to shed further light on the functions of PRP19-containing complexes in both normal and cancer cells.

POGZ promotes homology-directed DNA repair in an HP1-dependent manner.

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.

CUT Domains Stimulate Pol ß Enzymatic Activities to Accelerate Completion of Base Excision Repair.

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase ß to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol ß in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.

Ubiquitylation at the Fork: Making and Breaking Chains to Complete DNA Replication.

The complete and accurate replication of the genome is a crucial aspect of cell proliferation that is often perturbed during oncogenesis. Replication stress arising from a variety of obstacles to replication fork progression and processivity is an important contributor to genome destabilization. Accordingly, cells mount a complex response to this stress that allows the stabilization and restart of stalled replication forks and enables the full duplication of the genetic material. This response articulates itself on three important platforms, Replication Protein A/RPA-coated single-stranded DNA, the DNA polymerase processivity clamp PCNA and the FANCD2/I Fanconi Anemia complex. On these platforms, the recruitment, activation and release of a variety of genome maintenance factors is regulated by post-translational modifications including mono- and poly-ubiquitylation. Here, we review recent insights into the control of replication fork stability and restart by the ubiquitin system during replication stress with a particular focus on human cells. We highlight the roles of E3 ubiquitin ligases, ubiquitin readers and deubiquitylases that provide the required flexibility at stalled forks to select the optimal restart pathways and rescue genome stability during stressful conditions.

Cellular Depletion of BRD8 Causes p53-Dependent Apoptosis and Induces a DNA Damage Response in Non-Stressed Cells.

Regulation of the chromatin state is crucial for biological processes such as the regulation of transcription, DNA replication, and DNA damage repair. Here we show that knockdown of the BRD8 bromodomain protein - a subunit of the p400/Tip60 complex - leads to p21 induction, and concomitant cell cycle arrest in G1/S. We further demonstrate that the p53 transcriptional pathway is activated in BRD8-depleted cells, and this accounts for upregulation of not only p21 but also of pro-apoptotic genes, leading to subsequent apoptosis. Importantly, the DNA damage response (DDR) is induced upon BRD8 depletion, and DNA damage foci are detectable in BRD8-depleted cells under normal growth conditions. Consistently with an activated DDR, we find that in BRD8-depleted cells, the ATM-CHK2 DDR pathway is turned on but, CHK1 proteins levels are severely reduced and replication stress is detectable as enhanced replication protein A (RPA32) phosphorylation levels. Notably, acetylation of histone H4 at K16 (H4K16ac) is reduced in BRD8-depleted cells, suggesting that BRD8 may have a role in the recruitment and/or stabilization of the p400/Tip60 complex within chromatin, thereby facilitating DNA repair. Taken together, our results suggest that BRD8 is involved not only in p53-dependent gene suppression, but also in the maintenance of genome stability.

SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice.

DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair.

A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination.

RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.

SMARCAL1 Resolves Replication Stress at ALT Telomeres.

Cancer cells overcome replicative senescence by exploiting mechanisms of telomere elongation, a process often accomplished by reactivation of the enzyme telomerase. However, a subset of cancer cells lack telomerase activity and rely on the alternative lengthening of telomeres (ALT) pathway, a recombination-based mechanism of telomere elongation. Although the mechanisms regulating ALT are not fully defined, chronic replication stress at telomeres might prime these fragile regions for recombination. Here, we demonstrate that the replication stress response protein SMARCAL1 is a critical regulator of ALT activity. SMARCAL1 associates with ALT telomeres to resolve replication stress and ensure telomere stability. In the absence of SMARCAL1, persistently stalled replication forks at ALT telomeres deteriorate into DNA double-strand breaks promoting the formation of chromosome fusions. Our studies not only define a role for SMARCAL1 in ALT telomere maintenance, but also demonstrate that resolution of replication stress is a crucial step in the ALT mechanism.

Two distinct modes of ATR activation orchestrated by Rad17 and Nbs1.

The ATM- and Rad3-related (ATR) kinase is a master regulator of the DNA damage response, yet how ATR is activated toward different substrates is still poorly understood. Here, we show that ATR phosphorylates Chk1 and RPA32 through distinct mechanisms at replication-associated DNA double-stranded breaks (DSBs). In contrast to the rapid phosphorylation of Chk1, RPA32 is progressively phosphorylated by ATR at Ser33 during DSB resection prior to the phosphorylation of Ser4/Ser8 by DNA-PKcs. Surprisingly, despite its reliance on ATR and TopBP1, substantial RPA32 Ser33 phosphorylation occurs in a Rad17-independent but Nbs1-dependent manner in vivo and in vitro. Importantly, the role of Nbs1 in RPA32 phosphorylation can be separated from ATM activation and DSB resection, and it is dependent upon the interaction of Nbs1 with RPA. An Nbs1 mutant that is unable to bind RPA fails to support proper recovery of collapsed replication forks, suggesting that the Nbs1-mediated mode of ATR activation is important for the repair of replication-associated DSBs.