The essential role of RNase H1 in the generation and clearance of RNA primers for mitochondrial DNA replication

• Main Author: Holmes, J. B.
• Published: University of Cambridge 2011
• Subjects: 572.85
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604184

An approach was made to enrich isolated mitochondrial replication intermediates (mtRIs) for intact RNA/DNA hybrids. The S9.6 antibody preferentially binds RNA/DNA hybrids over other nucleic acid substrates, and so it was applied to highly purified preparations of mtDNA. mtRIs bound by the antibody were found to be enriched for RNase H sensitive mtRIs with blocked restriction sites, which are hallmarks of the RITOLS mechanism of replication. Conversely, mtRIs not bound by the antibody were RNase H resistant and cleaved on all branches by REs, consistent with products of strand-coupled DNA synthesis. Therefore, the S9.6 antibody based separation of mtRIs substantiates the earlier proposal of two classes of mtRI. To determine the role of RNase H1 <i>in vivo</i>, <i>Rnaseh1</i> conditional knockout mice were generated and mouse embryo fibroblasts (MEFs) cultured. The addition of tamoxifen to the culture media results in both <i>Rnaseh1</i> alleles being excised. In other experiments, wildtype and mutant versions of human RNase H1 were over-expressed in HEK293 cells. Mitochondrial DNA derived from these varied sources was analyzed by 1D-AGE, 2D-AGE, ligation mediated PCR (LM-PCR) and ribonuclease protection assay (RPA). Compared to control samples, the analysis found persistent RNA primers attached to the 5’ ends of nascent H- and L-strand molecules, combined with altered RNA-DNA transition sites. These nascent strands were sometimes incorporated into daughter molecules and ligated to upstream nascent DNA. These molecules were particularly unstable during subsequent rounds of replication and ultimately the mtDNA copy number was depleted upon modulation of RNase H1 levels. These data suggest that RNase H1 plays essential roles in generating and clearing RNAs that act as primers of DNA replication. RNase H1 primer processing is needed not only for initiation of replication, but also late in the replication cycle.