Aicardi-Goutières Syndrome (AGS) is a single gene, autoimmune disorder, with variable onset in the first year of life. Its clinical features exhibit similarities to several autoimmune diseases and congenital viral infections. AGS can result from mutations in ADAR1, TREX1 and SAMHD1 as well as any of the three genes that encode the protein subunits of the RNase H2 enzyme. It is hypothesised that impairment of nucleic acid metabolism results in abnormal nucleic acid species within the cell. This in turn is thought to cause the aberrant immune response that leads to AGS. The RNase H2 complex contains the catalytic RNASEH2A subunit and the auxiliary RNASEH2B and RNASEH2C subunits, which are thought to provide structural support and facilitate interactions with additional cellular proteins. RNase H2 can cleave the RNA strand of an RNA:DNA hybrid as well as 5’ of a single ribonucleotide embedded in dsDNA. Therefore, RNase H2 may have roles in several cellular processes, including DNA replication and repair, transcription, and viral infection. The aim of this PhD project was to investigate the physiological functions of RNase H2. The localisation of the RNase H2 proteins was investigated using EGFP-tagging and fluorescent microscopy. The interaction between the PIP-box of RNASEH2B and PCNA was found to localise RNase H2 and not RNase H1 to nuclear replication foci during S-phase. This suggests that RNase H2 is the dominant RNase H activity during DNA replication. Stable cell lines expressing EGFP-RNASEH2B and an alternative isoform, EGFP-RNASEH2Balt, were generated and used to perform a protein-protein interaction screen by GFP-Trap and mass spectrometry. The results indicate putative physical interactions between RNASEH2B and other factors involved in DNA replication and repair. Further evidence for a role in DNA repair was revealed when mammalian RNase H2 null cells were treated with hydroxyurea. Low doses of hydroxyurea increased ribonucleotide incorporation into genomic DNA and impaired S-phase progression. In contrast to wild-type cells, RNase H2 null cell proliferation also failed to recover from this replicative stress after HU withdrawal. However, the ribonucleotide content of genomic DNA from these cells did return to pre-hydroxyurea treatment levels. This suggests that an alternative repair pathway exists in mammalian cells, which can remove ribonucleotides from DNA in the absence of RNase H2, but that this pathway is also harmful to the cells. There is evidence that TREX1 facilitates viral infection while SAMHD1 has been shown to restrict viral infection. Therefore, experiments were performed to investigate if RNase H2 could be a viral facilitator or restriction factor. Ribonucleotides can be incorporated into viral DNA, so RNase H2 could act as a restriction factor by nicking and damaging the pre-integration complex. However, RNase H2 could also function as a facilitator of infection by processing viral RNA:DNA hybrid by-products and thus prevent the host immune response. The data obtained during this PhD project provides further evidence that RNase H2 is involved in DNA replication and repair and has contributed to the understanding of the function of RNase H2 in the cell. However, it is still unknown how mutations in RNase H2 lead to the pathology of AGS.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:693615 |
| Date | January 2014 |
| Creators | Rachel Astell, Katherine Rachel |
| Contributors | Jackson, Andrew ; Abbott, Catherine |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/16470 |
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