The proposed project has 3 Aims.
Aim 1. Determine whether origin firing in human cells is stochastic or deterministic; map the origin firing sites at high resolution; and identify functionally important origin motifs.
Using mouse embryonic stem cells (ESCs) we demonstrated that origin firing in early replicating genomic domains is deterministic, whereas in late replicating genomic domains it may be either stochastic or deterministic. Further, we mapped DNA replication origins at high resolution in human cells in tissue culture and, importantly, also in normal hepatocytes of mouse regenerating livers in vivo. Finally, we identified a sequence motif within DNA replication origins that is bound by the OCT4 pluripotency factor and which motif is required for firing of specific origins in mouse embryonic stem cells. An unexpected discovery made while studying origin firing in vivo in mouse regenerating livers was that old mice exhibited an origin firing defect and that this defect was due to DNA damage that had accumulated in the hepatocytes during aging, but had not been repaired.
Aim 2. Determine the mechanisms by which transcription in G1 suppresses firing of intragenic origins.
We explored two mechanisms: origin licensing occurs only in early G1 and then transcription removes any licensed origins within transcribed genes or transcription modifies chromatin in a way that prevents firing of licensed origins. Our results support the second mechanism, as we found that origin licensing requires Cdk4 activity, which is a late event in G1. These observations change how we think about origin licensing and firing, as they show that origin licensing requires Cdk activity, whereas previously it was thought (based on experiments in yeast) that Cdk activity inhibited origin licensing.
Aim 3. Determine why forks from intergenic origins do not collapse when replicating highly transcribed genes.
We determined the mechanism by which impending transcription-replication conflicts (TRCs) are sensed by cells. We found that TRCs are sensed by PARP1, which becomes activated and associates with Timeless, a protein that is part of the replication machinery. The interaction between activated PARP1 and Timeless pauses the DNA replication machinery, thereby averting collisions with the transcription machinery and the induction of DNA double-strand breaks and genomic instability. These findings explain how PARP inhibitors exert their therapeutic effect in the context of cancers that have defects in repair of DNA double-strand breaks, notably hereditary breast and ovarian cancers.
In the context of this Project we have published eleven papers. Two of these have been published in the most prestigious scientific journals: Nature and Cell, the remaining have been published in highly respected journals, including Nature Communications and Cell Reports. In addition, we have presented our findings in more than 15 international scientific meetings over the course of the past six years.
