Studying essential DNA metabolism proteins in Xenopus egg extract
Published: 13 October 2016
Vincenzo Sannino, Arun M. Kolinjivadi, Giorgio Baldi and Vincenzo Costanzo*
DNA metabolism Laboratory, IFOM, The FIRC institute of Molecular Oncology, Milan, Italy
The correct duplication of genetic information is essential to maintain genome stability, which is lost in cancer cells. Replication fork integrity is ensured by a number of DNA metabolism proteins that assist replication of chromatin regions difficult to replicate due to their intrinsic DNA sequence composition, coordinate repair of DNA molecules resulting from aberrant replication events or protect replication forks in the presence of lesions impairing their progression. Some DNA metabolism genes involved in DNA repair are essential in higher eukaryotes even in unchallenged conditions, suggesting the existence of biological processes requiring these specialized functions in organisms with complex genomes. The impact on cell survival of null mutants of many DNA metabolism genes has precluded complete in depth analysis of their function. Cell free extracts represent a fundamental tool to overcome survival issues. The Xenopus laevis egg cell free extract is an ideal system to study replication-associated functions of essential genes. We are taking advantage of this system together with innovative imaging and proteomic based experimental approaches to characterize the molecular function of essential DNA metabolism proteins. Using this approach we have uncovered the role of some essential homologous recombination and fork protection proteins in chromosomal DNA replication and we have characterized some of the factors required for faithful replication of specific vertebrate genomic regions. This approach will be instrumental to study the molecular mechanisms underlying the function of a number of essential DNA metabolism proteins involved in the maintenance of genome stability in complex genomes.
Xenopus laevis, immuno-depletion, DNA replication, DNA recombination and reversed forks