Institut Jacques Monod

Cadoret Lab

Pathology of DNA Replication

Jean-Charles CADORET

Genetic instability is a major characteristic of tumor cells and is associated with the process of carcinogenesis in humans. In many cases, such instability occurs during S-phase and is due to defects in DNA replication following replicative stress.

The team is pursuing three lines of research:

  • Project 1: Dynamics of the replisome; characterization of DNA polymerase pause sites during replication in the human genome, role of Pif1 Helicase and impact on genome stability.
  • Project 2: Impact of PARylation on replication control and genome stability
  • Project 3: Analysis of the temporal replication program and collaborations.

Keywords: DNA replication, Replicative stress, genomic stability

 +33 (0)1 57 27 80 73   Contact     @DNA_RepPath_lab

Within the team, we are developing and coordinating 3 areas of research and one area of collaboration:

Project 1: Replisome dynamics: Characterization of DNA polymerase pause sites during replication in the human genome, role of Pif1 helicase and impact on genome stability.

Our work aims to identify the molecular mechanisms of replication fork breaks, using human cells. This work should enable us to better understand how these replication defects, known as fork stoppers, can contribute to the development of cancers associated with genomic instability. The question now is whether replication pauses are due to: i) either stochastic events, i.e. they occur at location X at time Y and can vary from one cell cycle to another, or ii) they take place at a specific location which, if coordinated with other molecular processes, would enable the genome to be organized and thus reduce the risk of genetic instability.

To carry out this mapping of pause sites, we postulate that loci that impede the progression of replication forks show a longer association with the replisome compared with sites where there are no pauses. These break sites must therefore present a strong association with replicative DNA polymerases. We can therefore observe the distribution of DNA polymerase delta across the genome in replicating asynchronous cells using NGS approaches (ChIP-seq approach with Pol Delta antibody).

Profile obtained from sequencing with anti-pol epsilon and delta antibodies and from the Input sample

We are also using NGS and biochemical approaches to determine the role of the Pif1 helicase during replication and the impact of deregulation of this enzyme on genome stability. During the S-phase, replication forks may encounter several types of structures on chromatin that can impede fork progression. These obstacles can have several origins, for example: a) the formation of a DNA secondary structure, b) the formation of a DNA-Protein(s) complex c) a chromatin modification and/or finally d) transcription itself. Helicases of the Pif1 family are present in virtually all eukaryotes. Pif1 is required for mitochondrial DNA stability. It also appears to be involved in the resolution of specific structures such as G-quadruplexes (in vitro experiment). Finally, Rrm3 (homologous to Pif1) is known in S. cerevisae to facilitate fork progression across obstacles. Conflicts with transcription can have two origins: i) the association of transcription proteins with chromatin, or ii) the formation of transcription-derived RNA-DNA hybrids known as R-loops. The aim of this research project is therefore to characterize the role of Pif1 in the replication process and its impact on genome stability. In other words, we’d like to know whether the absence or overexpression of Pif1 induces replicative stress. In the human K562 line, we have obtained Pif1 KO clones using CRISP/Cas9 approaches, as well as a Pif1 overexpression line using PiggyBAC approaches, in which an HA tag has been added to the protein. On these two lines, we have initiated an initial experiment to compare the timing of replication.

Examples of replication timing profiles obtained for the Pif1 KO line and the Pif1 Surexpression line. In blue, the WT line, in red the genetically modified line. Green lines indicate an advance in timing for the mutant in the regions concerned, pink lines indicate delays.

We can already observe that the absence or overexpression of Pif1 deregulates replication timing. These effects are exacerbated by creating additional replicative stress in the presence of Aphidicolin. Finally, we observed a slowdown in cell growth for these different lines.

Project 2: Impact of CDK4-CDK6 inhibitors on replication and consequent genome stability

Palbociclib is a promising therapeutic molecule in the fight against cancer, as it belongs to the family of “CDK4 and 6 inhibitors”. However, resistance to these molecules can occur in cancer patients. So-called “pRb-negative” cancers are considered resistant to these molecules: cells continue to proliferate despite treatment. However, we have recently shown that these cells continue to proliferate, but have greater difficulty in carrying out DNA replication. This molecule therefore induces “replicative stress”. We are now trying to understand at what level this molecule interferes with the replication process. Using molecular combing approaches, we have observed a reduction in the number of active replication origins, but also an acceleration in the rate of replisome synthesis. This treatment has an effect on the molecular composition of the Initiation Precomplex of replication origins.

Project 3: Impact of Parylation on replication control

PARylation, via the PARP1 enzyme and NAD+, is the molecular process by which a multi-branched chain of ADP-Ribose (PAR) is synthesized at the protein (post-translational modification) or DNA level. This poly-ADP-ribose chain enables the recruitment of other proteins possessing PAR-binding peptide motifs. PARylation may thus be involved in several molecular processes such as chromatin remodeling, transcription, DNA repair and the cell cycle. PARP1’s involvement in DNA repair is the best described today. Briefly, PARP1 acts as a break detector, as it can interact with DNA thanks to the two zinc finger motifs this protein possesses in one of its domains. With this interaction, PARP1 then synthesizes a PAR chain, enabling the subsequent recruitment of proteins involved in DNA repair to the break site.

The involvement of PARP1 in DNA replication is very partially established, but the associated molecular mechanisms are not yet clear. It has been observed, in earlier work using fluorescence microscopy approaches (Coll et al., 1997), that PARP1 appears to be co-localized at sites of DNA synthesis but also at the initiation of this synthesis during the S phase of the cell cycle. Other work seems to show the presence of PARP1 on arrested replisomes (Ronson et al., 2018). We performed experiments in which cells were treated with PARPi or PARGi, inhibitors of the PARP and PARG enzymes respectively. We observed that cells have a strongly disrupted replication timing. EdU incorporation kinetics were performed to determine the effect of this type of treatment on DNA synthesis and replicative stress proteins. A decrease in EdU incorporation was observed at the second hour of treatment. With regard to replicative stress proteins, we once again observed, from the second hour, the appearance of phosphorylation of ChK1, a major protein in the response to replicative stress. Taken together, these preliminary data clearly indicate the direct role of PARylation in the replication process.

PARP inhibition by Olaparib and PARG inhibition by a specific inhibitor have an impact on the replication timing program in K562 cells. (left) Example of a replication timing profile visualized via START-R. The grey dots correspond to the probes, each covering a region of human chromosome 16 (hg18). The y-axis indicates early replication (positive values) and late replication (negative values). (right)Analysis of differences in replication timing between the conditions indicated. The number of regions with delayed or advanced replication is also indicated.

Project 4: Analysis of the temporal program of replication and collaborations.

We have created an App-Shiny to study the temporal program of replication throughout the genome (doi: 10.5281/zenodo.3243339 and https://thomasdenecker.github.io/START-R/index.html ).

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