Identification of novel factors regulating vertebrate DNA replication

DNA replication is a highly orchestrated process that ensures fidelity of genome during duplication. Despite extensive years of study the organization of the eukaryote replication machinery remains still elusive. the discovery of the molecular circuitry underlying the replication process could lead to know new pathways that could be potential new therapeutic targets to kill highly replicating cancer cells. To achieve this, we used cell-free systems derived from X. laevis eggs to identify novel protein complexes assembled on replicating chromatin. Recently, it has been shown that somatic nuclei are replicated very poorly when incubated in an interphase Xenopus egg extract. However, they can be reprogrammed to adapt to an embryonic DNA replication programme if pre-incubated in a mitotic arrested extract and a single mitosis is allowed. After this pre-incubation they become as efficient in DNA replication as embryonic nuclei and the inter-origin distance is converted from a somatic embryonic nuclei and the inter-origin distance is converted from a somatic distribution, one every 120kb, to an embryonic distribution, one every 10-15kb. On the basis of these findings, we fractionated the egg extract to isolate factors that influence activation of replication origins on somatic erythrocyte templates using differential PEG precipitation. Among all fractions we took one with a high rate of replication and one with a low rate of replication and we analysed them by Mass Spectrometry. In the fraction with a high efficiency of replication, sequencing by MS led to the identification of several factors involved in chromatin remodelling. We focused our studies a factor that we showed to have the highest ability to promote DNA replication of somatic nuclei when expressed as recombinant protein and incubated in egg extract. We named this factor SETRAF (Somatic to Embryonic TRAnsforming Factor). This on-going project will shed further light on the organization of the replication machinery, allowing a greater understanding of eukaryotic DNA replication.