Higher eukaryotes evolved with mechanisms that initiate DNA replication at multiple origins on multiple chromosomes. Activation of the replicative helicase at a single origin in each of ~50,000 replicons is sufficient to replicate the human genome in the absence of stress. These ~50,000 origins are selected from a five- to tenfold excess of licensed origins. Activation of additional replicative helicases at origins that would otherwise be passively replicated is observed after stress. This plasticity in origin use is a simple mechanism to recover DNA replication between stalled and collapsed replication forks. The mechanism(s) that limits origin firing to one per replicon in the absence of stress is not known. We recently reported that the DNA damage signaling kinases ATR and Chk1 inhibit activation of the replicative helicase in the absence of stress. In preliminary studies, we show that Chk1 kinase activity is strictly associated with ATR kinase-dependent phosphorylations on Chk1 and that these have an astonishingly short half-life in cells. We propose that this is a highly innovative mechanism that localizes Chk1 kinase activity to the immediate vicinity of ATR at active replicative helicases. We also show that Rif1, which has been implicated in the regulation of replication timing previously, is phosphorylated and that phosphorylated Rif1 binds protein phosphatase 1? (PP1?). Based upon these findings, we hypothesize that Chk1 kinase activity generates a ring of Rif1-PP1? around active replicative helicases and that this limits Cdc7 kinase-dependent origin firing across a replicon.
In Aim 1 we will investigate a new mechanistic paradigm for localizing DNA damage signaling to a small volume of the nucleus in the absence of stress.
In Aim 2 we will investigate the molecular mechanism that limits activation of the replicative helicase across a replicon in the absence of stress.
In Aim 3 we will investigate the impact of ATR kinase inhibition and conformal radiation on immune responses in tumor bearing mice. These studies are highly impactful as they will identify a fundamental mechanism that determines inter-origin distance, genome stability and the rate of cell division in higher eukaryotes. Since this mechanism may be attenuated in T cells, these studies will provide fundamental insights into adaptive immune responses. Our studies may have an immediate impact as the ATR and Chk1 kinase inhibitors used here are in clinical trials.
We will identify a fundamental mechanism that determines the distance between origins of DNA replication in human cells. This mechanism is a key determinant of genome stability and the rate of cell division, and accordingly it is a key determinant of therapeutic response in the medical and radiation oncology clinic.