We will determine how checkpoints regulate the cell cycle in response to damaged or incompletely replicated DNA during Drosophila development. Studies in cell extracts and unicellular systems have identified checkpoint mechanisms that monitor genomic DNA and modulate cell cycle progression, allowing time for repair and replication. During a metazoan life cycle, checkpoints must operate in the context of profound developmental changes in cell cycle regulation such as the abolishment of gap phases or mitosis. We are using Drosophila melanogaster to determine how known checkpoint mechanisms function during organismal developmental in vivo, and to uncover novel mechanisms not yet seen in other systems. Our initial findings indicate that different mechanisms act to stall mitosis in response to damaged DNA at different stages of Drosophila embryogenesis. The differential use of multiple mechanisms in metazoan development suggests that loss of a regulatory mechanism is more detrimental for one cell type than for another. This simple notion may help us to understand the tissue-specificity of human diseases that result from the loss of a checkpoint or a cell cycle regulator, and thus to design better therapies. Our plans include 4 aims. Aim 1) We have found that ongoing S phase is inhibited in response to DNA damage by ionizing radiation in a tissue/cell cycle specific manner in the larvae. Mutants in 01/S regulators (e.g Rb and E2F), G2/M regulators (e.g. Cdk1 and mitotic cyclins) and DNA repair proteins will be used to assay the role of these proteins in the inhibition of S phase, and to determine why only certain cells show this response. Aim 2) We have found that irradiation inhibited not only the entry into mitosis but also the progress through mitosis in Drosophila embryos; prophase becomes lengthened and metaphase anaphase transition becomes delayed. A combination of molecular, genetic and cytological approaches will identify mechanisms that regulate progress through mitosis after irradiation. Aim 3) We have found that incomplete DNA replication blocks the exit from mitosis via a spindle checkpoint protein and ultimately leads to apoptosis. A combination of genetic and cytological approaches, together with live imaging of chromosomes and mitotic spindles, will be used to uncover mechanisms that link incomplete DNA replication to the spindle checkpoint and to cell death. Aim 4) We will screen through existing mutagensensitive Drosophila mutants for their ability to regulate mitosis, S phase and cell death after irradiation, in order to uncover novel genes needed for proper response to DNA damage.

R01GM066441

2002-05-01

2008-04-30