ECOLE DOCTORALE/PHD PROGRAM CANCEROLOGY/ONCOLOGY SUJET DE THESE N° 49 ANNEE UNIVERSITAIRE 2014-2015 TITRE DU PROJET DE RECHERCHE (en français ET en anglais) Rôle de l’ADN polymerase zeta dans la réplication de régions d’ADN « difficiles à répliquer » et dans la mutagenèse spontanée. Determining the role of the DNA polymerase zeta in the replication of DNA regions "difficult to replicate" and in spontaneous mutagenesis. L’EQUIPE D’ACCUEIL DES DOCTORANTS Nom du directeur ou de la directrice de thèse (HDR requise) : Dr. Patricia Kannouche L’Equipe d’Accueil des Doctorants (Intitulé du Laboratoire, adresse postale, e-mail, téléphone) Unit of Genetic Stability and Oncogenesis UMR8200 - CNRS Institut Gustave Roussy - PR2 114, rue Edouard Vaillant 94805 - VILLEJUIF cedex E-mail : [email protected] Tél.: +33 1 42 11 40 30/ 51 18 Nom du directeur ou de la directrice du Laboratoire : Dr. Patricia Kannouche NOMBRE DE DOCTORANTS ACTUELLEMENT DANS L’EQUIPE D’ACCUEIL DES DOCTORANTS (nom, prénom et année d’inscription en thèse) Ahmed-Seghir Sana (2011, soutenance prévue en octobre 2014) Delrieu Noémie (2012) Ecole Doctorale de Cancérologie, Biologie, Médecine et Santé 418 DESCRIPTION DU PROJET DE RECHERCHE (en français ET en anglais) During cellular division the genome must be faithfully replicated to ensure an unaltered genomic transmission. Different origins along eukaryotic chromosomes are activated in early, middle or late S phase. This temporal control of DNA replication is referred to as the replication-timing program (1-2). Aberrant replication timing is observed in many different genetic diseases and it is associated with altered gene expression, impaired histone marks, mutagenesis and genomic instability. One prominent disease characterized by replication-timing aberrations is cancer (3-5). Cancer is a disease mainly caused by an accumulation of somatic mutations in normal cells, including singlenucleotide substitutions (SNS), small insertions, small deletions, amplifications and deletions of large genomic regions, and chromosomal translocations. How such alterations occur during DNA replication and influence the tumorigenesis remains elusive. Thanks to the development of NGS technologies and the sequencing of multiple cancer genomes, it appears that mutagenic events are not distributed randomly in the genome. A growing body of evidence indicates that the replication-timing program can influence the spatial distribution of mutagenic events such that certain regions of the genome which are replicated in late S phase present a strong increased spontaneous mutagenesis (SNS) compared to surrounding regions replicated in early S phase. In addition, these elevated mutation rates are associated with heterochromatin-like domains (6). One obvious question is how and why elevated mutation rates are associated with late replication in human cells. Heterochromatin constitutes a large portion of the eukaryotic genome. It is frequently located at the periphery of the nucleus and associated with centromeric and telomeric regions. Heterochromatin is mainly composed of long stretches of repetitive tandem DNA associated to specific repressive marks (e.g. DNA methylation and specific repressive histone marks) and is packaged into a less accessible form. In mammals, heterochromatin is replicated during the late S phase. So far, it remains unclear how the replication machinery can proceed through such barriers. In our lab, we are focusing on a special class of DNA polymerases (TLS polymerases) that support replication directly past template lesions (or unusual DNA secondary structure) that cannot be negotiated by the replicative high-fidelity polymerases. However, these specialized polymerases can be highly error-prone on undamaged DNA (for review, see (7)). Emerging concept proposes that these enzymes may also function during the unchallenged S phase (8-10). Among the TLS polymerases, the DNA polymerase zeta (Pol ) has been extensively characterized in the yeast S.cerevisiae. Its core complex is a heterodimer of the catalytic subunit Rev3 and its accessory subunit Rev7. One important feature is that most spontaneous and induced mutagenesis is dependent on Rev3 gene product in yeast. It has been recently reported that Pol also binds to the Pol31 and Pol32 subunits of Poldelta, forming a highly stable four-subunit called Pol (4) complex (Rev3-Rev7-Pol31-Pol32) which is structurally very close to the replicative DNA polymerase, Poldelta (11-12). However, its role remains unclear. In mammals Pol is unique among the TLS polymerases because it is the only one showing embryonic lethality in the mouse after inactivation of REV3L suggesting that this specialized polymerase has acquired an essential function during evolution which remains undetermined. On the basis of our preliminary data, we think that Pol can be required to replicate through condensed chromatin regions. The aim of the project is to decipher the role of the Polζ(4) complex during DNA replication in mammalian cells. To this end, mouse embryonic fibroblasts REV3+/+ and REV3-/- are available and the candidate will take advantage of a combination of powerful new technologies (DNA combing, iPOND, ChIP-seq) that are currently implemented in the laboratory. Using these techniques, the candidate will investigate the impact of Polζ(4) on the DNA Replication-timing and fork velocity when heterochromatin regions are replicated. In addition, he/(she) will map the Rev3 sites in the whole genome during replication and examine if the DNA binding sites of Rev3 correlate with constitutive and/or facultative heterochromatin. The candidate will then monitor the recruitment of Polζ(4) at the replication forks and identify its associated partners. Finally, mutational landscape in the genome of MEF REV3+/+ and REV3-/- will be examined. In conclusion, this project aims at uncovering novel aspects on DNA replication involving the Polζ(4) complex and we believe that it will shed new light on the mechanisms required to replicate through natural barriers such as heterochromatin, and might explain the elevated mutation rates associated with heterochromatin-like domains References 1. I. Hiratani et al., PLoS Biol 6, e245 (Oct 7, 2008). 2. D. M. Gilbert et al., Cold Spring Harb Symp Quant Biol 75, 143 (2010). 3. Y. Watanabe et al., Hum Mol Genet 11, 13 (Jan 1, 2002). 4. T. Ryba et al., Genome Res 22, 1833 (Oct, 2012). 5. A. C. Barbosa, P. A. Otto, A. M. Vianna-Morgante, Chromosome Res 8, 645 (2000). 6. B. Schuster-Bockler, B. Lehner, Nature 488, 504 (Aug 23, 2012). 7. J. E. Sale, A. R. Lehmann, R. Woodgate, Nat Rev Mol Cell Biol 13, 141 (Mar, 2012). 8. R. Betous et al., Mol Carcinog 48, 369 (Apr, 2009). 9. P. Sarkies, C. Reams, L. J. Simpson, J. E. Sale, Mol Cell 40, 703 (Dec 10). 10. S. S. Lange, J. P. Wittschieben, R. D. Wood, Nucleic Acids Res 40, 4473 (May, 2012). 11. A. V. Makarova, J. L. Stodola, P. M. Burgers, Nucleic Acids Res, (Oct 12, 2012). 12. A. G. Baranovskiy et al., J Biol Chem 287, 17281 (May 18, 2012) Ecole Doctorale de Cancérologie, Biologie, Médecine et Santé 418