Abstract
Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex-forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.
Highlights
Substrate G4_5 mimics a quadruplex in the heavy strand of mtDNA that spans nucleotides 581–525 (Fig. 6), which includes the HSP1 and houses one end of a 3,895-bp deletion first identified by Moraes et al in 1991 [41]
The deletion was flanked by 13-bp direct repeat sequences and resulted in loss of both rRNA sequences and the HSP1 at the first deletion breakpoint (Fig. 6A)
The frequency of occurrence of the 3,895-bp deletion was found to show a significant correlation with increasing UV exposure in samples collected from human skin [43]
Summary
Substrate G4_5 mimics a quadruplex in the heavy strand of mtDNA that spans nucleotides 581–525 (Fig. 6), which includes the HSP1 and houses one end of a 3,895-bp deletion first identified by Moraes et al in 1991 [41]. More than a decade later in 2004, Krishnan et al [43, 57] discovered that incidence of this large deletion was strongly correlated with UV exposure in human patients. The formation of this deletion could be triggered in cell culture through exposure to UV radiation. Ensuing studies have found that this deletion is present in human corneal stroma and macular regions of the retina
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