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.
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
(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
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
I. Hiratani et al., PLoS Biol 6, e245 (Oct 7, 2008).
D. M. Gilbert et al., Cold Spring Harb Symp Quant Biol 75, 143 (2010).
Y. Watanabe et al., Hum Mol Genet 11, 13 (Jan 1, 2002).
T. Ryba et al., Genome Res 22, 1833 (Oct, 2012).
A. C. Barbosa, P. A. Otto, A. M. Vianna-Morgante, Chromosome Res 8, 645 (2000).
B. Schuster-Bockler, B. Lehner, Nature 488, 504 (Aug 23, 2012).
J. E. Sale, A. R. Lehmann, R. Woodgate, Nat Rev Mol Cell Biol 13, 141 (Mar, 2012).
R. Betous et al., Mol Carcinog 48, 369 (Apr, 2009).
P. Sarkies, C. Reams, L. J. Simpson, J. E. Sale, Mol Cell 40, 703 (Dec 10).
S. S. Lange, J. P. Wittschieben, R. D. Wood, Nucleic Acids Res 40, 4473 (May, 2012).
A. V. Makarova, J. L. Stodola, P. M. Burgers, Nucleic Acids Res, (Oct 12, 2012).
A. G. Baranovskiy et al., J Biol Chem 287, 17281 (May 18, 2012)
Ecole Doctorale de Cancérologie, Biologie, Médecine et Santé 418