Exploring the impact of Replication Stress on Ageing and Cancer

DNA damage accumulates in our cells during life and it is a common cause for cancer and ageing. Accordingly, several human diseases (and mouse models) with mutations in genes related to genome maintenance display premature ageing and/or predisposition to cancer. However, whereas the role of DNA damage in human disease is well established, the specific impact of the different sources of DNA damage is less understood. To date, most research on DNA damage has focused on the responses to radiation (UV or ionizing radiation), reactive oxygen species (ROS), and telomere erosion. More recently, however, emphasis has shifted to the analysis of an intrinsic source of genome instability that arises from difficulties at the replication fork and that is known as replication stress (RS).

photo-Visualization of Replicative Stress in mammalian cells

Fig 1. Visualization of Replicative Stress in mammalian cells. Cells experiencing replication stress can be identified by the presence of a pan-nuclear phosphorylation of the histone H2A variant H2AX. Here, mouse embryonic fibroblasts were exposed to hydroxyurea, a reagent that depletes the pool of available dNTPs, and therefore stalls replication forks. In our lab, we use extensive High-Throughput microscopy for unbiased quantitative analysis of replication stress and other forms of DNA damage.

In short, replication stress is defined by the accumulation of large patches of single stranded DNA at stalled replication forks, which due to its recombinogenic nature has been proposed to be the main source of recurrent genomic rearrangements in cancer (reviewed in Lopez-Contreras and Fernandez-Capetillo, DNA Repair, 2010). In mammals, RS is sensed and suppressed by a signaling network coordinated by ATR and CHK1 kinases. In what relates to ageing, my previous laboratory (led by Dr Oscar Fernandez-Capetillo at CNIO, Madrid) discovered that replication stress can accelerate ageing in mammals (Murga et al., Nat Gen, 2009; Murga et al., NSMB, 2011).  These results revealed that a mammalian organism exposed to supraphysiological loads of replication stress ages faster than an unchallenged one, however the actual contribution of replication stress to normal ageing remains to be elucidated.

In my lab, I want to address the mechanisms by which replication stress influences ageing by taking advantage of a panel of mouse models that are naturally protected from RS. This would include, for instance, mice with increased levels of the CHK1 kinase –SuperChk1- (Lopez-Contreras et al., J Exp Med, 2012), or mice with an increased activity of the nucleotide production machinery –SuperRRM2- (Lopez-Contreras et al., unpublished). We already know that any of these strains can extend the lifespan of ATR mutant progeroid mice (Fig 1). However, whether these mice have an extended lifespan and better protection against age-related tissular decline remains to be addressed. The development of this project will also involve the use of state of the art Proteomic approaches, High-Throughput Microscopy and CRISPR/Cas9 technology, among others.

figure - RS-related mouse models of ageing

Fig 2. RS-related mouse models of ageing. A) Mice with reduced levels of the ATR kinase (ATR-Seckel) accumulate RS and age prematurely. B) When crossed with mouse models that are protected from RS (SuperChk1 or SuperRRM2) the lifespan of ATR mutant mice can be extended.

This project is financed by the Danish Council for Independent Research “Sapere Aude – DFF Starting Grant” (DFF – 4004-00185B)

Andres J Lopez-Contreras (Group Leader)
Alexandra Avram (Lab technician
Mauro Sbroggió (postdoc) 
Eliene Albers (PhD student)

Dr. Oscar Fernandez-Capetillo, Spanish National Cancer Research Center (CNIO), Madrid, Spain
Prof. Ian D. Hickson, Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark
Prof. Rafael Peñafiel-Garcia, University of Murcia, Spain
Dr. Javier Muñoz, Spanish National Cancer Research Center (CNIO), Madrid, Spain
Dr. Andre Nussenzweig, National Cancer Institute, National Institutes of Health, Bethesda, USA
Dr. Jeremy A. Daniel, Center for Protein Research, University of Copenhagen, Denmark.