Eukaryotic DNA replication starts at multiple sites throughout the genome and is necessarily coordinated with transcription, sister chromatid cohesion, nucleosome assembly and cell cycle progression. In addition to the complexity of the replication reaction it, during replication cells need to deal with DNA damage and stalled forks, originated inevitably by the action of exogenous and endogenous agents. The success of this process is crucial to preserve genome stability, and the inability to deal with DNA lesions during replication or to protect or restart stalled forks leads to DNA breaks, chromosomal rearrangements, and mutations that can cause the loss of cell viability, but in addition errors in DNA replication result in a large number of human syndromes, including premature aging, various cancer predispositions and genetic abnormalities. To solve or reduce these problems, cells use repair and detoxification pathways as well as surveillance mechanisms, called checkpoints, which serve to detect the problem and coordinate repair with chromosome segregation and progression through the cell cycle.
Most genes involved in cell cycle regulation, DNA replication, repair and genome stability, are largely conserved in all organisms, including yeast, animals and plants. Examples of them are the members of the pre-replication complex (pre-RC), such as ORC, CDC6, CDT1, or MCM, or components of the DNA damage signaling pathways, such as ATM or ATR, just to cite a few. Structural genes for DNA replication (polymerases, MCM, ORC, PCNA, etc.), DNA repair (RAD3/XPG; CSB/RAD26, MSH2, etc.), double-strand break repair and recombination (Rad51, Rad54, Sgs1-BLM, BRCA2, Ku70-Ku80, MR(X)N complex, etc.), SMC proteins (cohesins, condensins, etc.), and checkpoint proteins (Rad9, Rad24, ATM-ATR, Rad53, etc.) are good examples of proteins essential for the maintenance of genomic instability with a key role in cell cycle, all of which are conserved from eukaryotic microorganisms such as yeast, to humans. Understanding the function of these genes and proteins as well as the mechanisms in which they participate is essential not only in Basic and Fundamental research but in Biomedicine. Thus, mutations in many of these genes constitute the molecular basis of different human diseases, including cancer. For example, retinoblastoma, Bloom syndrome, DiGeorge syndrome, Ataxia telangiectasia, Xedorerma pigmentosum, Cockayne's syndrome, Nijmegen Break syndrome, Werner syndrome, Non-polyposic hereditary colon cancer, breast cancer, among many others, arise as a consequence of loss-of-function mutations in key cell cycle and DNA replication and repair genes. For an integral view of how these processes, which include cell cycle regulation, DNA replication, DNA repair and recombination, checkpoint mechanisms and chromosomal segregation, function in a coordinated manner to warrant genome integrity we need to know their proteins and mechanisms.
Figure 1:
A general view of DNA insults and consequences on cell cycle and DNA repair
For an integral view of how these processes function in a coordinated manner to safeguard genome integrity and how they can impact cell viability and proliferation we are making a coordinated effort devoted to:
- Identify and decipher the mechanisms of cell cycle regulation at the onset of DNA replication;
- decipher the eukaryotic factors and mechanisms of initiation of DNA replication;
- determine the mechanisms of action of DNA polymerases and replication fork progression;
- identify the factors, functions and mechanisms controlling DNA repair and recombination,
- Identify the role of cohesion in DNA repair and chromosomal segregation.
We use a complementary and varied multiplicity of approaches, including Biochemistry, Cell Biology, Genetics, Molecular Biology, Genomics and Proteomics, in different model organisms including phage ø29, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, Xenopus egg cell-free systems, mouse embryonic fibroblasts and different human cell lines as well as mice, the ultimate goal of the project being the deciphering of the mechanisms that prevent genomic instability.