ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage–induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage–induced checkpoint activation and translesion synthesis in mammalian cells

Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA

Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin

Influence of the live cell DNA marker DRAQ5 on chromatin-associated processes

In the last decade, live cell fluorescence microscopy experiments have revolutionized cellular and molecular biology, enabling the localization of proteins within cellular compartments to be analysed and to determine kinetic parameters of enzymatic reactions in living nuclei to be measured. Recently, in vivo DNA labelling by DNA-stains such as DRAQ5, has provided the opportunity to measure kinetic reactions of GFP-fused proteins in targeted areas of the nucleus with different chromatin compaction levels. To verify the suitability of combining DRAQ5-staining with protein dynamic measurements, we have tested the cellular consequences of DRAQ5 DNA intercalation. We show that DRAQ5 intercalation rapidly modifies both the localization and the mobility properties of several DNA-binding proteins such as histones, DNA repair, replication and transcription factors, by stimulating a release of these proteins from their substrate. Most importantly, the effect of DRAQ5 on the mobility of essential cellular enzymes results in a potent inhibition of the corresponding cellular functions. From these observations, we suggest that great caution must be used when interpreting live cell data obtained using DRAQ5

Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle

DNA translesion synthesis (TLS) is a crucial damage tolerance pathway that oversees the completion of DNA replication in the presence of DNA damage. TLS polymerases are capable of bypassing a distorted template but they are generally considered inaccurate and they need to be tightly regulated. We have previously shown that polη is phosphorylated on Serine 601 after DNA damage and we have demonstrated that this modification is important for efficient damage bypass. Here we report that polη is also phosphorylated by CDK2, in the absence of damage, in a cell cycle-dependent manner and we identify serine 687 as an important residue targeted by the kinase. We discover that phosphorylation on serine 687 regulates the stability of the polymerase during the cell cycle, allowing it to accumulate in late S and G2 when productive TLS is critical for cell survival. Furthermore, we show that alongside the phosphorylation of S601, the phosphorylation of S687 and S510, S512 and/or S514 are important for damage bypass and cell survival after UV irradiation. Taken together our results provide new insights into how cells can, at different times, modulate DNA TLS for improved cell survival

Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate

Osteogenesis imperfecta (OI) types VII, VIII and IX, caused by recessive mutations in cartilage associated protein (CRTAP), prolyl-3-hydroxylase 1 (P3H1), and cyclophilin B (CyPB), respectively, are characterized by the synthesis of overmodified collagen. The genes encode for the components of the endoplasmic reticulum (ER) complex responsible for the 3-hydroxylation of specific proline residues in collagen type I. Our study dissects the effects of mutations in the proteins of the complex on cellular homeostasis, using primary fibroblasts from seven recessive OI patients. In all cell lines the intracellular retention of overmodified type I collagen molecules causes ER enlargement associated to the presence of protein aggregates, activation of the PERK branch of the unfolded protein response and apoptotic death. The administration of 4-phenylbutyrate (4-PBA) alleviates cellular stress by restoring ER cisternae size, normalizing the p-PERK/PERK ratio and the expression of apoptotic marker. The drug has also a stimulatory effect on autophagy. We proved that the rescue of cellular homeostasis following 4-PBA treatment is associated to its chaperone activity, since it increases protein secretion, restoring ER proteostasis and reducing PERK activation and cell survival also in presence of autophagy pharmacological inhibition.Our results provide a novel insight into the mechanism of 4-PBA action and demonstrated that the intracellular stress in recessive OI can be tuned by 4-PBA therapy, similarly to what we recently reported for dominant OI, thus allowing a common target for OI forms characterized by overmodified collagen

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.This work was supported by funding from the Medical Research Council and the European Research Council (ERC, 281847) (A.P.J.), the Lister Institute for Preventative Medicine (A.P.J. and G.S.S.), Medical Research Scotland (L.S.B.), German Federal Ministry of Education and Research (BMBF, 01GM1404) and E-RARE network EuroMicro (B.W), Wellcome Trust (M. Hurles), CMMC (P.N.), Cancer Research UK (C17183/A13030) (G.S.S. and M.R.H), Swiss National Science Foundation (P2ZHP3_158709) (O.M.), AIRC (12710) and ERC/EU FP7 (CIG_303806) (S.S.), Cancer Research UK (C6/A11224) and ERC/EU FP7 (HEALTH-F2- 2010-259893) (A.N.B. and S.P.J.).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.345

The Regulation of DNA Damage Tolerance by Ubiquitin and Ubiquitin-Like Modifiers

DNA replication is an extremely complex process that needs to be executed in a highly accurate manner in order to propagate the genome. This task requires the coordination of a number of enzymatic activities and it is incredibly fragile and prone to arrest after DNA damage. DNA damage tolerance provides a last line of defense that allows completion of DNA replication in presence of an unrepaired template. One of such mechanisms is called Post Replication Repair (PRR) and it is used by the cells to bypass highly distorted templates caused by damaged bases. PRR is extremely important for the cellular life and performs the bypass of the damage both in an error free and in an error prone manner. In light of these two possible outcomes, PRR needs to be tightly regulated and controlled in order to prevent accumulation of mutations leading ultimately to genome instability. Post-translational modifications provide the framework for this regulation and Ubiquitylation and SUMOylation of PRR proteins play a pivotal role in choosing which pathway to activate, controlling the different outcomes of damage bypass. PCNA (Proliferating Cell Nuclear Antigen), the DNA clamp for replicative polymerases, plays a central role in the regulation of damage tolerance and its modification by Ubiquitin and SUMO controls both the error free and error prone branches of PRR. Furthermore, a significant number of polymerases involved in the bypass of DNA damage possess domains that can bind post-translational modifications and they are themselves target for ubiquitylation. In this review, we will focus on how Ubiquitin and Ubiquitin-like modifications can regulate the DNA damage tolerance systems and how they are capable of controlling the recruitment of different proteins to the replication fork

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end joining

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ