(483.4) Unique DNA Polymerase kappa Interactome Suggests Novel Cellular Functions

Poster Board Number: A4

Shilpi Paul (Penn State University College of Medicine), Paolo Cifani (Cold Spring Harbor Laboratory), Darryl Pappin (Cold Spring Harbor Laboratory), Thomas Spratt (Penn State University College of Medicine)

Translesion DNA synthesis (TLS) polymerases evolved to tolerate DNA damage that bypasses DNA lesions, thus preventing genomic instability. Multiple TLS polymerases exist with different damage tolerance capabilities, since they have low fidelity their access to the replication fork must be regulated to minimize mutations. The current paradigm is that a combination of kinetic partitioning and protein-protein interactions are used to regulate TLS polymerase activity. A major knowledge-gap in elucidating the roles of these polymerases is that it is difficult to identify which polymerase is active in a specific situation. We designed and synthesized a novel nucleotide analog N2-benzyl- 2′-deoxyguanosine (EBndG) that is highly selective toward DNA polymerase kappa (Pol κ), a Y family TLS polymerase. Pol k can bypass bulky lesions in the minor groove generated by a metabolite benzo[a]pyrene diolepoxide (BPDE), an environmental carcinogen. Although Pol κ has been identified to have multiple cellular roles, the mechanisms regulating its different cellular activities are unknown.

            To interrogate the identity of proteins surrounding the Pol κ active sites, we performed an extensive study using modified iPOND (isolation of proteins on nascent DNA), called iPoKD (isolation of proteins on Pol kappa synthesized DNA). Human cell lines were treated with BPDE, subsequently 5-ethynyl-2-deoxyuridine (EdU) or EBndG was added and proteins bound to the DNA containing EdU and EBndG were analyzed by mass spectrometry. In addition, we performed quantitative analysis of the Pol κ active sites interactome using iPoKD followed by iTRAQ (isobaric tags for relative and absolute quantitation). Our data identified DNA replication and repair proteins previously identified with EdU pull-downs; and interestingly enrichment of RNA binding, ribosome biogenesis, nucleolar proteins and transcriptional repressive complex(es) associated with EBndG pull-downs. Chromatin modifiers, histone chaperones and histone variants are identified suggesting changes in chromatin structure that facilitates Pol κ-mediated DNA lesion bypass, repair and restorative process. identification of unique proteins associated with EBndG-containing DNA, suggests novel roles for Pol κ’s activity in the cell.

            Using super-resolution confocal microscopy, Pol κ activity is identified in the nucleolus after BPDE damage. EBndG is observed in the nucleolar DNA and Pol κ s activity regulated by the canonical polycomb-complex recruited by the PARylation of PARP1. BPDE lead to transcriptional stress and repression that is gradually recovered. We are investigating whether Pol κ maintains ribosomal DNA integrity after BPDE damage and is required for TLS DNA synthesis or repair. Pol κ active site associated candidate proteins are being validated using CRISPR-Cas9 knockout or siRNA knockdown strategies. These data will provide first insight into Pol κ’s core interactome, its regulation, chromatin surrounding Pol κ active sites and its novel cellular roles.

Funding: R01ES021762-07(TS), NIH Re-Entry into Biomedical Research(SP)