Causes and Consequences of DNA Replication Stress in the Mammalian Preimplantation Embryo

Project Summary/Abstract The genome of the early embryo provides the genetic information of the adult and thereby determines the health of a person. Surprisingly, though an intact genome is paramount to normal development, the genome of some mammalian species is highly unstable, including in human. Cleavage-stage embryos acquire high levels of DNA damage and frequent chromosomal abnormalities that adversely affect developmental potential. We recently showed that the key reason for these abnormalities are high levels of DNA replication stress during the first cell cycles. The causes of replication stress are not known, and we are only beginning to understand the consequences. Therefore, there is a strong rationale to study DNA replication in the mammalian embryo. A hypothesis central to this proposal is that DNA replication stress is caused by epigenetic remodeling during the first cell cycles. We furthermore hypothesize that the dynamics of DNA replication, also called DNA replication timing, predispose specific genomic regions to instability and chromosome breakage. We will test this hypothesis in three specific Aims. In Aim1, we will identify the causes of DNA replication stress. In Aim2, we will characterize replication timing during preimplantation development, identify its molecular determinants in the preimplantation embryo, and correlate these with the sites of chromosome breakage determined in Aim3. The proposed work is innovative, because it focuses on an essential process - the replication of the DNA - that has thus far not been examined as a developmentally relevant factor in mammals, and which has not yet been investigated as a source of genetic change. It is also innovative because of the use of animal model systems which accurately reproduce the defects seen in human cells. The contribution is significant, because the proposed studies will lead to an understanding of the causes, mechanisms, and consequences of genome instability in the first few cell divisions of mammalian development. The work proposed will help understand the source of both normal as well as disease-causing genetic variation in the genome.