The TIMELESS Roles in Genome Stability and Beyond.

TIMELESS protein (TIM) protects replication forks from stalling at difficult-to-replicate regions and plays an important role in DNA damage response, including checkpoint signaling, protection of stalled replication forks and DNA repair. Loss of TIM causes severe replication stress, while its overexpression is common in various types of cancer, providing protection from DNA damage and resistance to chemotherapy. Although TIM has mostly been studied for its part in replication stress response, its additional roles in supporting genome stability and a wide variety of other cellular pathways are gradually coming to light. This review discusses the diverse functions of TIM and its orthologs in healthy and cancer cells, open questions, and potential future directions.

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells.

DNA polymerase epsilon (PolE) in an enzyme essential for DNA replication. Deficiencies and mutations in PolE cause severe developmental abnormalities and cancers. Paradoxically, the catalytic domain of yeast PolE catalytic subunit is dispensable for survival, and its non-catalytic essential function is linked with replicative helicase (CMG) assembly. Less is known about the PolE role in replication initiation in human cells. Here we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in U2OS cells. POLE1-depleted cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 non-catalytic domain rescued this defect resulting in slow, but continuous DNA synthesis. We propose a model where in human U2OS cells POLE1/POLE2 are dispensable for CMG assembly, but essential during later steps of replication initiation. Our study provides some insights into the role of PolE in replication initiation in human cells.

Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/ß expression.

ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. ATR kinase inhibitors (ATRi's) combine with radiation to generate CD8+ T cell-dependent responses in mouse models of cancer. We show that ATRi's induce cyclin-dependent kinase 1 (CDK1)-dependent origin firing across active replicons in CD8+ T cells activated ex vivo while simultaneously decreasing the activity of rate-limiting enzymes for nucleotide biosynthesis. These pleiotropic effects of ATRi induce deoxyuridine (dU) contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and interferon-α/ß (IFN-α/ß). Remarkably, thymidine rescues ATRi-induced dU contamination and partially rescues death and IFN-α/ß expression in proliferating CD8+ T cells. Thymidine also partially rescues ATRi-induced cancer cell death. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell-dependent anti-tumor responses in mouse models. We conclude that ATR is essential to limit dU contamination in genomic DNA and IFN-α/ß expression.

WEE1 kinase inhibitor AZD1775 induces CDK1 kinase-dependent origin firing in unperturbed G1- and S-phase cells.

WEE1 kinase is a key regulator of the G2/M transition. The WEE1 kinase inhibitor AZD1775 (WEE1i) induces origin firing in replicating cells. We show that WEE1i induces CDK1-dependent RIF1 phosphorylation and CDK2- and CDC7-dependent activation of the replicative helicase. WEE1 suppresses CDK1 and CDK2 kinase activities to regulate the G1/S transition after the origin licensing is complete. We identify a role for WEE1 in cell cycle regulation and important effects of AZD1775, which is in clinical trials.

The human Shu complex functions with PDS5B and SPIDR to promote homologous recombination.

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.

Dormant origin signaling during unperturbed replication.

Mechanisms that limit origin firing are essential as the ˜50,000 origins that replicate the human genome in unperturbed cells are chosen from an excess of ˜500,000 licensed origins. Computational models of the spatiotemporal pattern of replication foci assume that origins fire stochastically with a domino-like progression that places later firing origins near recent fired origins. These stochastic models of origin firing require dormant origin signaling that inhibits origin firing and suppresses licensed origins for passive replication at a distance of ∼7-120 kbp around replication forks. ATR and CHK1 kinase inhibitors increase origin firing and increase origin density in unperturbed cells. Thus, basal ATR and CHK1 kinase-dependent dormant origin signaling inhibits origin firing and there appear to be two thresholds of ATR kinase signaling. A minority of ATR molecules are activated for ATR and CHK1 kinase-dependent dormant origin signaling and this is essential for DNA replication in unperturbed cells. A majority of ATR molecules are activated for ATR and CHK1 kinase-dependent checkpoint signaling in cells treated with DNA damaging agents that target replication forks. Since ATR and CHK1 kinase inhibitors increase origin firing and this is associated with fork stalling and extensive regions of single-stranded DNA, they are DNA damaging agents. Accordingly, the sequence of administration of ATR and CHK1 kinase inhibitors and DNA damaging agents may impact the DNA damage induced by the combination and the efficacy of cell killing by the combination.

An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication.

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

Regulation of the initiation of DNA replication in human cells.

The origin of species would not have been possible without high fidelity DNA replication and complex genomes evolved with mechanisms that control the initiation of DNA replication at multiple origins on multiple chromosomes such that the genome is duplicated once and only once. The mechanisms that control the assembly and activation of the replicative helicase and the initiation of DNA replication in yeast and Xenopus egg extract systems have been identified and reviewed [1,2]. The goal of this review is to organize currently available data on the mechanisms that control the initiation of DNA replication in human cells.

Human DNA polymerase ε is phosphorylated at serine-1940 after DNA damage and interacts with the iron-sulfur complex chaperones CIAO1 and MMS19.

We describe a dynamic phosphorylation on serine-1940 of the catalytic subunit of human Pol ε, POLE1, following DNA damage. We also describe novel interactions between POLE1 and the iron-sulfur cluster assembly complex CIA proteins CIAO1 and MMS19. We show that serine-1940 is essential for the interaction between POLE1 and MMS19, but not POLE1 and CIAO1. No defect in either proliferation or survival was identified when POLE1 serine-1940 was mutated to alanine in human cells, even following treatment with DNA damaging agents. We conclude that serine-1940 phosphorylation and the interaction between serine-1940 and MMS19 are not essential functions in the C terminal domain of the catalytic subunit of DNA polymerase ε.

DNA damage-induced ubiquitylation of proteasome controls its proteolytic activity.

Stability of proteins is largely controlled by post-translational covalent modifications. Among those, ubiquitylation plays a central role as it marks the proteins for proteasome-dependent degradation. Proteolytic activities of proteasomes are critical for execution of various cellular processes, including DNA damage signaling and repair. However, very little is known about the regulation of proteasomal activity in cells during genotoxic stress. Here we investigated post-translational modifications of the 20S proteasomal subunits upon DNA damage induced by doxorubicin. Using mass-spectrometry, we found novel sites of phosphorylation and ubiquitylation in multiple proteasome subunits upon doxorubicin treatment. Ectopic co-expression of proteasome subunits and tagged ubiquitin confirmed the presence of ubiquitylated forms of PSMA5, PSMA1, PSMA3 and PSMB5 in cells. Moreover, we demonstrated that ubiquitylation in vitro inhibited chymotrypsin-like and caspase-like activities of proteasomes. In vivo, doxorubicin increased the activity of proteasomes, paralleling with attenuation of the overall level of proteasome ubiquitylation. Collectively, our results suggest a novel mechanism whereby the proteolytic activities of proteasomes are dynamically regulated by ubiquitylation upon DNA damage.

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism.

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli.

26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo- RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (alpha5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (alpha6) and PSMA3 (alpha7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.