Description of the PhD thesis project
proliferation depends on the faithful and accurate duplication of the genome.
The integrity of the genome is as its most vulnerable during DNA replication.
The DNA replication machinery is continuously threatened by a broad spectrum of
unavoidable fork obstacles such as DNA damage causing fork stalling and
collapse, a phenomenon known as replication stress (RS) (Zeman, NCB 2014).
Failure to safeguard genome stability upon RS is a potent driving force behind
the onset and progression of cancer cells. Cells have evolved a range of
factors, known as the “DNA Damage Response” (DDR) to protect the genome by
repairing DNA lesions and promoting the completion of DNA replication.
Understanding the underlying DDR mechanisms is an important aspect of cancer
biology, as DDR dysfunction is linked to cancer development and several DDR
factors are potent therapeutic targets, especially to target exacerbated RS in
fork processing usually relies on homologous recombination for error-free repair.
However, recent works indicates that non-homologous end joining (NHEJ) is
unexpectedly active during DNA replication to ensure fork protection, restart
and repair (Audoynaud, TIGS 2021). The lab recently discovered a novel
RNA-based mechanism to regulate the function of the NHEJ-factor Ku in the
processing of stressed forks in fission yeast. This proposal aims at unraveling
how RNA species modulate human KU function during RS response.
Human Ku is part of the multistep mechanism of fork-degradation
(Dhoonmoon Nat Com. 2022). Ku binding to RNA:DNA hybrid at RS site is likely to
represent an underappreciated level of regulation yet crucial in fine-tuning
NHEJ events at stressed fork. To solve this, this interdisciplinary program
integrates state-of the art cell and molecular biology approaches associated to
biophysics methods to reveal the fundamental aspects of human Ku functions at
stressed forks and how RNA species may modulate these functions. Such
understanding may pave new avenues to define novel therapeutics approaches.
We will study:
- The molecular determinants of Ku binding to nascent
DNA in human cells:
i) the role of RNA:DNA hybrids and their origin,
type of stressed forks are bound by Ku using distinct RS inducing agents and
iii) the role of DDR kinases Such analysis will be performed in
established cell lines and extended to triple negative breast cancers models.
- The structural determinant of human Ku binds to
RNA:DNA hybrids and DNA with the aim to define separation-of-function mutants.
- The cell response to RS in separation-of-function
mutants to elucidate how RNA species modulate its function during RS.
International, interdisciplinary &
intersectoral aspects of the project
student will visit the “Single-Molecule Biophotonics research laboratory” (New
York, University School of Medicine) to be trained in cutting-edge single
molecule fluorescence imaging techniques and computational methods. Super
resolution microscopy allows single molecule detection with spatial resolution
tenfold improved over conventional confocal microscopy to track Ku at
replication fork (Whelan DR, Lee WTC, Marks F, Kong
YT, Yin Y, Rothenberg E. Super-resolution
visualization of distinct stalled and broken replication fork structures. PLoS
Genet. 2020 Dec 28;16(12):e1009256.).
student will benefit from mentoring and secondment by people working in private
sector will share and pass on his experience to the student, act as
accompanying tutor and participate to the yearly PhD committees.
The interdisciplinary of the proposal lies in
the complementary approaches used to reach the goals. In addition to receiving
training in molecular and cell biology, the student will be exposed to technics
and concepts at the frontiers of physics. Training in super resolution
microscopy will allow the student acquiring interdisciplinary skills in the
physics principles of cell imaging and computational methods for analysis. The
collaboration with JB. Charbonnier, who was pioneer to solve crystal and CryoEM
structures of human Ku-DNA complexes (Nemoz C,
Ropars V, Frit P, Gontier A, Drevet P, Yu J, Guerois R, Pitois A, Comte A,
Delteil C, Barboule N, Legrand P, Baconnais S, Yin Y, Tadi S, Barbet-Massin E,
Berger I, Le Cam E, Modesti M, Rothenberg E, Calsou P, Charbonnier JB. XLF and APLF bind Ku80 at two remote sites to
ensure DNA repair by non-homologous end joining. Nat
Struct Mol Biol. 2018 Oct;25(10):971-980.), the student will acquire skills in
state-of-the-art biochemistry (ITC, EMSA) and structural biology. Beyond the
technical and specific training, the student will be confronted with diverse
and interdisciplinary scientific cultures that will nurture his/her scientific
maturity and vision.
- Zeman MK, Cimprich KA. Causes and consequences of replication
stress. Nat Cell Biol. 2014 Jan;16(1):2-9.
- Audoynaud C, Vagner S, Lambert S. Non-homologous end-joining at challenged
replication forks: an RNA connection? Trends Genet. 2021 Nov;37(11):973-985.
- Charlotte Audoynaud, Anissia Ait
Saada, Paloma Fernández Varela, Armelle Gesnik, Virginie Boucherit, Virginie
Ropars, Karine Fréon, Jean Baptiste Charbonnier, Sarah Lambert. RNA:DNA Hybrids From Okazaki
Fragment are cis-Regulators of Ku Function in Safeguarding
Replication Fork Integrity. Under revision (https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4017909).
- The KU-PARP14 axis differentially regulates
DNA resection at stalled replication forks by MRE11 and EXO1. Dhoonmoon A, Nicolae CM, Moldovan GL. Nat Commun. 2022 Aug 27;13(1):5063.