Poster abstracts
Poster number 133 submitted by Niraja Soman
RNase H1-S233A mutant fails to suppress R loop associated genomic instability
Niraja Soman (Masonic cancer center, Department of Pharmacology, University of Minnesota), Hai Dang Nguyen (Masonic cancer center, Department of Pharmacology, University of Minnesota)
Abstract:
R loops are three stranded RNA:DNA hybrid structures having multiple biological applications in natural cellular processes. They play important roles in gene expression, DNA replication and repair. However, how the formation of unscheduled R loops causes genomic instability and its association with cancer is unknown. To avoid detrimental effects of R loops, they need to be tightly regulated. RNaseH1 is an enzyme that degrades the RNA strand in the RNA:DNA hybrid to resolve R loops. Yet, how RNaseH1 is regulated is not fully understood. Here, we found that RNaseH1 is phosphorylated at Ser76 and Ser233 in cells by phospho-proteomics. In this project, we focused on how RNase H1 phosphorylation at Ser233 plays a role in R-loop regulation. The Ser233 is located on the DNA binding channel in the catalytic domain (CAT) at a basic protrusion in a phosphate binding pocket, which is unique to human RNaseH1 protein (Nowotny et al., Mol Cell 2007). To investigate RNaseH1 function in cells, we generated doxycycline inducible GFP-tagged phospho-mutant (S233A) and phospho-mimetic (S233E) in U2OS cell lines. To assess R-loop-associated genomic instability, we treated cells with a PARP1/2 specific inhibitor, olaparib (PARPi), and measured DNA damage levels by immunofluorescence staining using anti-γH2AX. In contrast to RNaseH1WT-GFP overexpressing cells, neither individual RNaseH1S76A-GFP nor RNaseH1S233A-GFP mutants were able to suppress PARPi-induced DNA damage. Future studies will be conducted to investigate the molecular function of Ser233 phosphorylation on RNaseH1 activity.
References:
Yang, W., Lee, J. Y., & Nowotny, M. (2006). Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity. Molecular cell, 22(1), 5-13.
Keywords: RNase H1, Genomic instability, R loops