Regulation of DNA replication in Xenopus laevis

The purpose of my research project is to make an attempt to understand some aspects of eukaryotic DNA replication by studying how this process is regulated positively or negatively in different physiological conditions. Using Xenopus egg extracts as the in vitro replication system, I studied the inhibitors of DNA replication found in nuclei of Xenopus quiescent cells. Replication of nuclei isolated from quiescent Xenopus cells was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with about 100% efficiency in the same extracts. The evidence of the inhibitors came further from the fact that replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei. The formation of pre-replication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when the nuclear extracts of liver cells were added to metaphase egg extracts prior to or immediately after a Ca++ ion-induced transition to interphase. A plausible interpretation of the data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in both somatic and cultured Xenopus cells. This may be achieved by negatively regulating positive modulators of the replication machinery;One of the requirements for the inception of DNA synthesis is the unwinding of the DNA duplex for loading of replication-related factors. Single-stranded DNA within intact nuclei was identified by laser confocal microscopy after staining with the bis-intercalating dye YOYO-1. Ratiometric image analysis showed that chromosomal DNA in intact nuclei of cleaving Xenopus embryos (blastulae) and gastrulae was extensively single-stranded. In contrast, chromosomal DNA in non-dividing quiescent brain nuclei was nearly indistinguishable from control double-stranded DNA in intact sperm nuclei. Ratiometric image analysis also established that DNA in demembranated sperm nuclei incubated in Xenopus egg extracts became extensively single-stranded as replication proceeded in vitro. These observations constitute the strongest evidence to date for the single-stranded replicative intermediates predicted by the "Strand Separation Model" for chromosomal DNA replication.