Introduction and general objective
The pre-replication complex (pre-RC), assembled at DNA origins of replication, comprehends an origin recognition complex (ORC, containing Orc1-Orc6 plus Cdc6 and Cdt1 proteins) and the mini-chromosome maintenance complex (MCM, composed of Mcm2-Mcm7 proteins) .
Origins of replication are licensed by the preRC assembling, but DNA synthesis initiation requires additional proteins. The binding of regulatory factors and structural proteins allows DNA unwinding, recruitment of DNA polymerases and establishment of replication forks.
Eukaryotic genomes replicate during the S phase from many replication origins, which are licensed hours before in early G1 phase. On the other hand, ORC1-6, Cdc6, and Cdt1 are down-regulated by the end of G1 to avoid DNA rereplication during the same cell cycle . When cultured quiescent G0/G1-arrested mammalian cells are stimulated by serum factors to progress across G1 phase, DNA replication origins licensing and the subsequent down-regulation of the ORC are robustly coupled to cell growth by still poorly understood molecular mechanisms.
In fact, it has been reported that Cdc6 and Cdt1 over-expression and/or deregulation can lead to DNA re-replication, genetic instability, gene amplification, DNA damage response (DDR) and, eventually, cancer . To deal with this complexity our experimental approach is to focus on 2 sets of regulatory molecular switches:
a) senescence _ proliferation transition switches, in which we analyze FGF2 dual control of proliferation in the mouse Y1 adrenocortical malignant cell line;
b) DNA replication switches, in which we study pre-replication complex (pre-RC) and replication fork machinery of trypanosomes, which are evolutionarily ancient protozoan parasites.
Results will likely uncover novel molecular regulatory mechanisms to elaborate descriptive signaling networks underlying cell cycle control with original and local flavor, which is a major focus of the Center research.
In addition, it is of immediate therapeutic interest the elucidation of cellular and molecular bases of DNA replication control in trypanosomes, etiological agents of “Chagas” and “Sleeping Sickness” diseases.
References. 1. Bell & Dutta (2002) Ann Rev Biochem71:333; 2. Blow JJ & Gillespie PJ(2008) Nature Rev Cancer 8: 799; 3. Costa ET et al (2008) Cancer Res 68: 6215; 4. Godoy et al (2009) Eukaryot Cell 8: 1592.