Oral Presentation 28th Lorne Cancer Conference 2016

Dissecting substrate dependent mechanisms of Cdc7-mediated replication fork protection using chemical genetics and quantitative phosphoproteomics (#26)

Mathew JK Jones 1 , Stephanie Munk 2 , Amnon Koren 3 , Steven McCarroll 3 , Jesper Olsen 2 , Mark Frattini 4 , Prasad V Jallepalli 1
  1. Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
  2. University of Copenhagen and Novo Nordisk Foundation Center for Protein Research, Copenhagen, Denmark
  3. Harvard Medical School, Boston, MA, USA
  4. Division of Hematology/Oncology, Columbia University Medical Center, New York, NY, USA

The uncontrolled proliferation of cancer cells places a large amount of stress on the cellular machinery required for DNA replication. In response, cancer cells frequently overexpress replication components such as the cell-cycle kinase, Cdc7. Small molecule Cdc7 inhibitors have shown encouraging results in preclinical tumor models; however, their broad specificity and weak penetrance in vivo prohibit their ability to functionally assess the role of Cdc7.  Genetic systems for Cdc7 have revealed that the kinase performs a vital role in initiating DNA replication.  In contrast, little is known about Cdc7’s later functions and substrates after initiation when DNA synthesis is ongoing. To address these issues, we employed adeno-associated virus (AAV) vectors to delete and replace the Cdc7 gene (CDC7L1) in human somatic cells with a “shokat” allele that can be rapidly and specifically inhibited by small molecule bulky ATP analogs. Using this approach, we have discovered that Cdc7 is required to preserve the stability and restart potential of stalled replication forks.  To understand the mechanisms underlying this regulation, we compared “Cdc7-on” and “Cdc7-off” cells using SILAC and quantitative phosphoproteomics. We identified novel Cdc7-dependent phosphorylation sites on the cohesin ring complex and components of the breast cancer susceptibility complex BRCA1-A. Using CRISPR-mediated gene-editing to generate stable knockout cell lines for these substrates, we found that the Cdc7-phosphorylated forms of these substrates make complementary contributions to fork protection: whereas cohesin stabilizes nascent strands during the stall, the BRCA1-A complex promotes restart after the block to elongation is removed.  Our findings demonstrate a novel role for Cdc7 after initiation, at stalled replication forks.  Since replication fork stalling is elevated upon oncogenic transformation and chemotherapy treatments, these insights may be harnessed to improve clinical treatment options and optimize Cdc7 inhibition therapeutically.