The physiological level of rNMPs present in mtDNA does not compromise its stability

Abstract

MtDNA integrity is important for mitochondrial function. We analysed mtDNA over the course of mouse lifespan and found that its integrity, as measured by electrophoretic mobility under denaturing conditions, decreased significantly in old animals whereas its ribonucleotide (rNMP) content increased with age. To investigate whether the rNMPs incorporated into mtDNA were responsible for this loss of integrity in old animals, we used mice deficient in SAMHD1, a dNTPase that decreases rNTP/dNTP ratios. Knockout of SAMHD1 almost completely eliminated rNMPs from mtDNA, but did not restore mtDNA integrity in older animals, indicating that rNMP incorporation does not explain the loss of mtDNA integrity. Neither did mtDNA from older animals contain more gaps, deletions or double-strand breaks than that of younger animals. By contrast, mtDNA integrity was restored by treatment with ligase. Thus, we conclude that the loss of mtDNA integrity in old mice is caused by accumulation of nicks. The genome of mitochondria in mammalian cells is a circular 16 kb dsDNA molecule present in multiple copies per cell. This mitochondrial (mt)DNA encodes key subunits of the respiratory chain complexes that synthesize the majority of the cell's ATP. Consequently, a partial loss of the number of copies of mtDNA from the cell (i.e. depletion) or defects in the quality of the DNA can affect energy production and manifest as human disease [1]. MtDNA quality also decreases with age due to the progressive accumulation of deletions and point mutations [2][3][4], which may contribute to the physiology of aging [5].Ribonucleotides (rNMPs) incorporated into DNA are an important threat to genome stability. Although replicative DNA polymerases are highly selective for dNTPs, the large excess of rNTPs over dNTPs in the cell results in incorporation of some rNMPs during DNA replication [6,7]. Due to their reactive 2ʹ-hydroxyl group, these rNMPs increase the risk of strand breaks by several orders of magnitude [8]. Furthermore, the presence of rNMPs in DNA can alter its local structure and elasticity [9][10][11], thus interfering with processes such as replication, transcription or DNA repair. To prevent these negative effects of incorporated rNMPs on genome stability, they are removed from nuclear DNA (nDNA) by the ribonucleotide excision repair pathway, which is initiated by cleavage at the incorporated rNMP by RNase H2 [7,12]. RNase H2 is essential for genome stability in mammals and its absence results in increased single-stranded DNA (ssDNA) breaks and an activated DNA damage response [13,14].In contrast to nDNA, mtDNA contains persistent rNMPs that are incorporated during replication by the mtDNA polymerase, Pol ɣ (reviewed in [15]). Human Pol ɣ is highly selective against using rNTPs as substrates and thus incorporates fewer rNMPs than nuclear replicative DNA polymerases do [16][17][18]. Once incorporated, however, rNMPs persist in mtDNA because mitochondria lack repair pathways for their removal [19,20]. Although a certain level of rNMPs is tolerate...