Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Genesis
2017 Jan 01;551-2:. doi: 10.1002/dvg.22996.
Show Gene links
Show Anatomy links
Xenopus egg extract to study regulation of genome-wide and locus-specific DNA replication.
Raspelli E
,
Falbo L
,
Costanzo V
.
???displayArticle.abstract???
Faithful DNA replication, coupled with accurate repair of DNA damage, is essential to maintain genome stability and relies on different DNA metabolism genes. Many of these genes are involved in the assembly of replication origins, in the coordination of DNA repair to protect replication forks progression in the presence of DNA damage and in the replication of repetitive chromatin regions. Some DNA metabolism genes are essential in higher eukaryotes, suggesting the existence of specialized mechanisms of repair and replication in organisms with complex genomes. The impact on cell survival of many of these genes has so far precluded in depth molecular analysis of their function. The cell-free Xenopus laevis egg extract represents an ideal system to overcome survival issues and to facilitate the biochemical study of replication-associated functions of essential proteins in vertebrate organisms. Here, we will discuss how Xenopus egg extracts have been used to study cellular and molecular processes, such as DNA replication and DNA repair. In particular, we will focus on innovative imaging and proteomic-based experimental approaches to characterize the molecular function of a number of essential DNA metabolism factors involved in the duplication of complex vertebrate genomes.
Figure 1. Xenopus egg extract preparation. Collected eggs are activated with Calcium ionophore (middle) or left untreated (top), packed and crushed by low speed centrifugation. This separates the egg contents into a lipid plug floating on the top, an insoluble pellet on the bottom mainly composed by mitochondria, yolk and pigments, and a cytoplasmic layer in between (LSS), which is either mitotic or interphasic. An additional high-speed centrifugation of LSS allows the separation of membranes from the cytosolic fraction (HSS). Interphase LSS can also be used to prepare nucleoplasmic extract (bottom). For this purpose, sperm nuclei replicated in interphase LSS are collected by gentle centrifugation in the presence of microtubule depolymerizing agents, resulting in a nuclear layer floating on the top of the extract. Nucleoplasmic extract is then obtained by high-speed centrifugation of this nuclear layer
Figure 2.
Exposure to mitotic environment increases replication efficiency of somatic nuclei. Cartoon showing somatic nuclei incubated in LSS before (top) or after CaCl2 addition (bottom) to release extracts in interphase. Mitotic activities induce chromosome formation (middle tube), thereby allowing resetting of the replicon. This allows somatic nuclei pre-incubated in mitosis (top) to have a shorter inter origin distance compared to somatic nuclei incubated directly in interphase (bottom). This is reflected in a more efficient DNA replication
Figure 3.
Structural and regulatory features of centromeric DNA. Repetitive centromeric chromatin is organized in loops of dsDNA enriched for condensins (SMC2-4) and Topoisomerase I (Top I). Efficient DNA synthesis depends on MSH2-MSH6 proteins, which might be required to resolve the secondary structures arising on centromeric repeats. This topological arrangement limits the formation of extensive ssDNA regions and RPA hyper-loading onto chromatin, triggered by the uncoupling of helicase and polymerase induced by fork stalling agents. Inhibition of RPA binding to chromatin prevents ATR activation, resulting in Chk1 activity suppression and more efficient centromeric DNA replication
