Investigating PARP-mediated slowdown of Replication using scEdU-seq

Abstract

PARP enzymes are polymerases that facilitates the attachment of poly(ADP-ribose) chains to effector proteins, and are known for their ability to recognize and signal sites of DNA damage. Previously, we have shown that transcription-induced DNA damage slows down replication speeds. Furthermore, inhibition of PARP enzymes increases DNA replication speeds, however, which mechanisms link PARP signaling and DNA replication speeds is unknown. To explore these mechanisms, we generated inducible CRISPRi cell lines targeting known partners of PARP1 and explored their impact on replication through a variety of assays. We find that SSRP1 knockdown cells have increased rate of EdU incorporation in S-phase and display a reduction in cellular proliferation. Furthermore, SSRP1 knockdown cells showed a largely increased sensitivity to the inhibition of Chk1 kinase, but not the upstream ATR kinase. Finally, using the novel scEdU-seq method, we find that SSRP1-depleted cells display a sudden drop in replication speeds in late S-phase cells. Thus, SSRP1 regulates DNA replication speeds, which is likely through its role in histones eviction during late S-phase. Future research focused on understanding how cells coordinate the spatially-coinciding processes of transcription, DNA repair and -replication, will potentially generate insights on cancer, defective development disorders and immunodeficiencies.