Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE

Abstract

A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA.

Thermodynamic analysis of mitochondrial DNA breakpoints reveals mechanistic details of deletion mutagenesis

“…Indeed, TWINKLE overexpression in WT mice led to an increased level of mtDNA rearrangements (see Supplementary Figure S6 in the original publication 48 ). In addition to its putative function as a helicase, TWINKLE has been shown to catalyze DNA recombination 49,50 . A recent study further demonstrated that strand exchange activity of TWINKLE requires 3-6 bp sequence homology 50 The one exception to this pattern is the autosomal polg mutations.…”

“…In addition to its putative function as a helicase, TWINKLE has been shown to catalyze DNA recombination 49,50 . A recent study further demonstrated that strand exchange activity of TWINKLE requires 3-6 bp sequence homology 50 The one exception to this pattern is the autosomal polg mutations. POLG is the sole DNA polymerase present in mammalian mitochondria.…”

Mechanistic and biological considerations of oxidatively damaged DNA for helicase-dependent pathways of nucleic acid metabolism

“…Thus, Twinkle’s ability to upregulate mitochondrial biogenesis can help to alleviate oxidative stress associated with cardiac dysfunction. Twinkle’s ability to catalyze homologous strand exchange and DNA branch-migration and its proposed role in recombinational repair and remodeling of stalled mitochondrial replication forks [42, 208] may be relevant to situations of elevated oxidative stress.…”

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

“…To our knowledge, this and another study (34) published while ours was under revision are the first reported demonstrations that Twinkle possesses DNA branch migration activity of a D-loop, a DNA substrate the enzyme is likely to encounter during mtDNA replication. Collectively, the in vitro studies suggest that Twinkle may be capable of acting upon D-loop substrates by a combination of its branch migration and helicase activity, the latter not restricted to D-loops with pre-existing 5Ј singlestranded DNA tails.…”