Centre Nationale de Recherche Scientifique (CNRS) UMR8200, Laboratoire Stabilité Génétique et Oncogenèse, Université Paris-Sud, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France.
"Indeed, replication fork stalling at UV-induced DNA damage activates a checkpoint regulated by the ATR kinase that allows S-phase slow down and stabilization of replication forks (for review, see Cimprich & Cortez, 2008). We have previously shown that persistent inhibition of DNA synthesis in UV-irradiated cells deficient in Pol was associated with a stronger and persistent activation of ATR checkpoint (Despras et al, 2010). We analyzed the phosphorylation status of various ATR targets. "
"The identity of this band with Polη was demonstrated in wild-type cells transfected with a siRNA produced against the POLH gene. In this experiment, we used a unique siRNA because it has been already tested in our laboratory [Despras et al., 2010] and it is used only to show the disappearance of the protein band. As shown in Figure 2C, the 80 kDa band almost completely disappears in the siRNA-treated cells indicating we are looking to the real Polη protein. "
[Show abstract][Hide abstract] ABSTRACT: Xeroderma pigmentosum-variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XP-V patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, 6 for squamous-cell carcinoma and 5 for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure. This article is protected by copyright. All rights reserved.
Human Mutation 02/2014; 35(1). DOI:10.1002/humu.22462 · 5.14 Impact Factor
"In contrast, our data indicate that expression of the Cdt1 PIP degron on its own affects cell viability on UV damage, similar to full-length Cdt1, suggesting interference with TLS function, independently from Cdt1 function in DNA replication. There is evidence that replication fork restart at UV lesions requires Pol η (66), which may suggest that removal of Cdt1 from PCNA after UV damage in early S-phase may facilitate Pol η recruitment to reduce replication stress. This interpretation is also consistent with a previous report showing that Pol η is essential for cell viability on UV damage (15), and that disruption of its PCNA interaction region strongly affects cell viability (67). "
[Show abstract][Hide abstract] ABSTRACT: Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.
Nucleic Acids Research 01/2014; 42(6). DOI:10.1093/nar/gkt1400 · 9.11 Impact Factor
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