Molecular mechanisms that prevent DNA re-replication

Abstract

DNA replication must be precisely regulated to ensure faithful inheritance of genetic information. In mammalian cells, the process of genomic duplication starts from thousands of replication origins. During G1, the Origin Recognition Complex (ORC), CDC6 and CDT1 proteins cooperate to engage the MCM helicase complex at the origins. This process is called ‘origin licensing’ and renders cells competent to initiate DNA replication in the subsequent S phase. After the S phase starts, initiator proteins are inhibited to prevent origin re-licensing and reactivation. If this control is overridden, cells undergo aberrant DNA re-replication, which causes DNA damage, genomic instability and even cell death. The effects of DNA re-replication have been mainly studied in cells in culture, but not in mammalian organisms in vivo. In the first part of this Thesis we have studied the consequences of CDC6 and CDT1 deregulated expression in mice. We have identified a limiting role for CDC6 in origin licensing and activity. We have also found that both CDC6 and CDT1 need to be overexpressed in combination to induce re-replication in primary MEFs and adult tissues. Highly proliferative cells, including embryonic stem cells and hematopoietic precursors showed a marked susceptibility to CDT1 overexpression, which correlated with high expression of endogenous CDC6. In the mouse, DNA re-replication caused severe tissue dysplasias that become lethal in less than 2 weeks. In the second part of the Thesis we have carried out a genetic screening to find new mechanisms that control the extent of re-replication caused by origin refiring. We have identified FBH1 and RAD51 as members of a new pathway that limits this aberrant process. RAD51 binds to chromatin in S phase and acts as a molecular brake to hinder progression of re-replication forks. This role depends on the binding of RAD51 to DNA around the origins but is independent of homologous recombination. Finally, we have found that MRE11 nuclease might be also involved in this process by catalyzing the resection of re-replicated DNA.