Role of Replication in Activation of the DNA Damage Checkpoint

摘要

Current work in our lab has shown that a synthetic structure consisting of M13 ssDNA with one annealed oligonucteotide (called 1-80) is capable of activating the checkpoint in a replication independent manner, confirming our model that primed ssDNA is the checkpoint activating signal (see above). To determine if the amount of ssDNA following a primer affects the level of Chkl phosphorylation, an oligonucleotide will be annealed to a smaller ssDNA plasmid. This structure and the original structure will be added to Xenopus egg extract in equal amounts and the levels of Chkl phosphorylation will be compared. If ssDNA following a primer-template junction amplifies the checkpoint response, then the original M13 1-80 structure will yield higher levels of Chkl phosphorylation than the smaller plasmid. We are also developing a protocol for analyzing proteins bound to ssDNA using a peptide-nucleic acid (PNA) sequence. This will allow us to determine protein binding requirements during checkpoint activation using the 1-80 structure.