Molecular Oncology Programme

DNA Replication Group

Group Leader:  Juan Méndez
Research highlights
Genome-wide analysis of replication origin usage upon replicative stress

Mammalian DNA replication starts at tens of thousands of points called ‘origins’ whose frequency of activation is flexible. Our laboratory reported, several years ago, that stalled replication forks induce the activation of extra origins as a backup mechanism (Ibarra et al., Proc Natl Acad Sci USA, 2008). In collaboration with the group of Dr María Gómez (Centro de Biología Molecular “Severo Ochoa”, Madrid), we have used a technique called SNSSeq (isolation of short nascent DNA strands followed by DNA sequencing) to map the genomic positions and global efficiency of origins in mouse embryonic stem cells under experimental conditions that trigger extra origin activation. This approach allows a comparative analysis of ‘constitutive’ origins that are active in all the tested conditions, as well as the analysis of ‘responsive’ origins that become stimulated under stress. We have found that constitutive origins are more frequently associated with open chromatin marks, CpG islands, bivalent promoters and transcription factors than responsive ones. In addition, many replication origins increase their frequency of activation following stress. In collaboration with computational biologists at CNIO and the Barcelona Supercomputing Center, we are working on integrating the linear origin maps into threedimensional chromatin networks, in order to shed light on the complex hubs of nuclear DNA replication factories.

Lethal tissue dysplasias caused by DNA re-replication

Activation of oncogenes such as c-Myc affects replication origins through mechanisms that are not well understood. It has been reported that oncogenic stress may lead to origin hyperactivation, which leads to DNA over-replication and possibly gene amplification. Origin activity is largely controlled by the CDC6 and CDT1 proteins responsible for the loading of the MCM DNA helicase onto DNA. In normal cells, the activity of CDC6 and CDT1 is strictly regulated to prevent origin reactivation, but both factors are overexpressed in multiple types of cancer.

To investigate the effects of deregulated expression of CDC6 and CDT1 in vivo, we have designed mouse strains that allow the inducible expression of both proteins, alone or in combination. This year, we have reported that simultaneous, but not individual, deregulation of CDC6 and CDT1 is lethal to adult mice due to a striking dysplasia of the intestinal epithelium. The regeneration of intestinal epithelia is driven by transit-amplifying cells derived from stem cell niches located at the bottom of the intestinal crypts. Upon CDC6 and CDT1 deregulation, transit-amplifying progenitors undergo DNA over-replication and cell death, causing rapid intestinal failure (FIGURE).

A mechanistic insight derived from this study is that lethal DNA over-replication only occurred when CDC6 and CDT1 were deregulated at the same time. Therefore, unleashing CDT1 activity could kill tumour cells that generally express high levels of CDC6, but have no effect on the surrounding tissue that display normal CDC6 expression. At this time, CDT1 stimulation can be achieved by targeting GMN, a CDT1 inhibitor, or by the use of neddylation inhibitors (e.g. MLN4924) that stabilise CDT1. The mouse strains characterised in our study reveal the marked cytotoxicity of DNA re-replication in vivo and may therefore become useful models in pre-clinical studies.

PrimPol: a damage tolerance protein with potential applications in cancer therapy

We continue to investigate the functions of PrimPol, an enzyme that facilitates replication through damaged DNA. We have found that genetic ablation of PrimPol by Crispr/Cas9 sensitises cells to UV and chemical agents that induce DNA damage. Because PrimPol facilitates cell survival in the presence of DNA lesions, its inhibition could enhance the efficacy of cytotoxic chemotherapeutic agents that trigger cell death by inducing DNA damage.