Cells respond to DNA damage by activating a multi-faceted response called DNA damage response. Defects in this response can result in genomic instability, a major feature of cancer cells. Several cancer-prone syndromes reflect defects in the specific genes of the DNA damage response. They include Ataxia-Telangiectasia or A-T, Nijmegen Breakage Syndrome and A-T Like Disorder or A-TLD. Several players of the DNA damage response and DNA repair processes are known. However, their biochemical function is poorly understood. To understand the molecular function of these genes in their biological context we set up cell-free systems based on Xenopus laevis egg extracts that recapitulate the DNA damage responsein vitro and allow biochemical studies of its different steps. Extracts derived from Xenopus laevis eggs allow extensive biochemical analysis and can reproduce in vitro complex cell cycle events such as chromatin formation, nuclear assembly, semi-conservative DNA replication, chromosome assembly and mitotic spindle formation.
Using this system we identified novel ATM and ATR DNA damage checkpoints that prevent initiation of DNA replication following DNA damage. We studied DNA repair pathways and identified novel functions for the Mre11 complex that account for its essential role in vertebrates. We identified the mechanism involved in the Mre11 dependent chromosome break processing in Xenopus laevis and human cells. More recently, we have dissected the molecular mechanisms underlying Rad51 and Mre11 function during DNA replication using new methodologies that we developed based on Electron Microscopy (EM) to visualize replication intermediates derived from egg extract. We demonstrated that Mre11 promotes degradation of nascent strands at stalled forks and that Rad51 limits this processing. We have also shown that ATM regulates the pentose phosphate pathway promoting the formation of antioxidant molecules such as NADPH and nucleotide precursors required for DNA repair. Finally, we have isolated genes present exclusively in vertebrate organisms such as GEMC1 and Cep63 that regulate essential cell cycle transitions such as initiation of DNA replication and mitosis spindle assembly under perturbed and unchallenged conditions.
In contrast to lower eukaryotes many genes involved in DNA repair and in DNA damage response are lethal when inactivated in mammalian cells even in the absence of apparent DNA damage. This indicates that these genes fulfil essential tasks for cell survival in complex organisms. In addition, many genes controlling DNA metabolism such as BRCA1 and BRCA2, the Fanconi anemia proteins and p53 are only present in metazoan organisms implying a higher intrinsic complexity of DNA metabolism.
The ongoing projects of my group aim at understanding the molecular roles played by vertebrate DNA metabolism genes in various aspects of cell cycle, DNA replication and DNA repair in unchallenged and stressful conditions. In particular, we are analysing the coordination of DNA replication, DNA damage repair and DNA damage checkpoint at molecular level. We are dissecting the signal transduction pathways that sense DNA damage and promote cell cycle arrest and DNA damage repair. We are characterizing novel substrates of the ATM/ATR pathway and looking for small molecules that modulate DNA damage response and repair.
To this end, we are applying a multidisciplinary approach. In particular, we are taking advantage of cell-free extracts, mass-spectrometry analysis of protein-protein interaction networks, antibody based techniques, mammalian cell manipulation, mouse genetics and human diseases such as Ataxia-Telangiectasia as tools and model systems.
The high degree of genetic conservation between Xenopus and mammalian organisms facilitates the biochemical study of large essential proteins that can be easily isolated and characterised. An important tool available with this system is the possibility of depleting native proteins and protein complexes using specific antibodies. This approach allows the identification of the precise biochemical steps in which these proteins operate allowing the study of essential vertebrate genes that would normally compromise viability when inactivated in other cellular systems. This system offers unique opportunities not available with other known experimental models to define the molecular role of essential DNA metabolism proteins.
Figure: Experimental approaches
This approach is complemented by the use of mass spectrometry analysis of DNA repair, DNA replication and DNA damage response protein complexes immuno-purified from egg extract with specific antibodies to identify the complete set of partners of these protein complexes in defined biological contexts. We are also using new tools based on advanced imaging technologies such as EM analysis of DNA metabolism intermediates. Our findings are being elaborated in light of comparative studies with mammalian cells and integrated by metabolic and developmental analysis aimed at understanding the role of DNA metabolism genes in wider aspects of cellular and organism physiology. These studies will ultimately lead to the determination of biological and biochemical function of genes involved in essential cellular processes that are dysfunctional in cancer cells.
The role of essential DNA repair and DNA damage response genes in vertebrate DNA replication
(update: April 2015)
15 aprile 2015 rel.02