Faithful chromosomal DNA replication is essential to maintain genome stability. A number of DNA metabolism genes are
involved at different levels in DNA replication. These factors are thought to facilitate the establishment of replication origins,
assist the replication of chromatin regions with repetitive DNA, coordinate the repair of DNA molecules resulting from
aberrant DNA replication events or protect replication forks in the presence of DNA lesions that impair their progression.
Some DNA metabolism genes are present mainly in higher eukaryotes, suggesting the existence of more complex repair
and replication mechanisms in organisms with complex genomes. The impact on cell survival of many DNA metabolism
genes has so far precluded in depth molecular analysis. The use of cell free extracts able to recapitulate cell cycle events
might help overcoming survival issues and facilitate these studies. The Xenopus laevis egg cell free extract represents an
ideal system to study replication-associated functions of essential genes in vertebrate organisms. We will take advantage of
this system together with innovative imaging and proteomic based experimental approaches that we are currently
developing to characterize the molecular function of some essential DNA metabolism genes. In particular, we will
characterize DNA metabolism genes involved in the assembly and distribution of replication origins in vertebrate cells,
elucidate molecular mechanisms underlying the role of essential homologous recombination and fork protection proteins in
chromosomal DNA replication, and finally identify and characterize factors required for faithful replication of specific
vertebrate genomic regions.
The results of these studies will provide groundbreaking information on several aspects of vertebrate genome metabolism
and will allow long-awaited understanding of the function of a number of vertebrate essential DNA metabolism genes
involved in the duplication of large and complex genomes.