Structure–function defects of the twinkle amino-terminal region in progressive external ophthalmoplegia

Abstract

TWINKLE is a DNA helicase needed for mitochondrial DNA replication. In lower eukaryotes the protein also harbors a primase activity, which is lost from TWINKLE encoded by mammalian cells. Mutations in TWINKLE underlie autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with multiple deletions in the mtDNA. Four different adPEO-causing mutations (W315L, K319T, R334Q, and P335L) are located in the N-terminal domain of TWINKLE. The mutations cause a dramatic decrease in ATPase activity, which is partially overcome in the presence of single-stranded DNA. The mutated proteins have defects in DNA helicase activity and cannot support normal levels of DNA replication. To explain the phenotypes, we use a molecular model of TWINKLE based on sequence similarities with the phage T7 gene 4 protein. The four adPEO-causing mutations are located in a region required to bind single-stranded DNA. These mutations may therefore impair an essential element of the catalytic cycle in hexameric helicases, i.e. the interplay between single-stranded DNA binding and ATP hydrolysis.

Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity

“…Published reports that the W315L, K319E, R334Q, P335L, I367T, S369P, R374Q, and L381P p72 variants are deficient for DNA helicase function (13,24,29) also disagree with our results (Fig. 4C).…”

“…The demonstrated DNA helicase activities of the W315L, K319E, I367T, S369P, R374Q, and L381P variants (Fig. 4C) disagree with published reports that these amino acid substitutions significantly impair or even inactivate helicase function (13,24,29). A, the ability of WT p72 to unwind a forked oligonucleotide substrate was determined as described under "Experimental Procedures," except that reactions contained 3 (lanes 1-4), 6 (lanes 5-8), 12 (lanes 9 -12), or 20 nM (lanes 13-16) WT p72 hexamers.…”

“…4). Comparisons of specific activity are further complicated because the previous studies employed assays with a 30 -90-fold stoichiometric excess of p72 hexamers over the DNA substrate (16,24,25,29). Although excess enzyme would facilitate the formation of sufficient enzyme-DNA complex to produce a detectable helicase signal, we found the addition of surplus enzyme to be counterproductive, if not inhibitory to helicase function (see supplemental Fig.…”

“…Although ϳ4-fold variability in helicase function was observed within the mutant collection, none of the p72 variants displayed a profound defect in helicase activity. In contrast, published DNA helicase assays utilized only 0.075-0.33 nM oligonucleotide substrate (13,16,24,25,29), conditions under which unwinding activity is directly proportional to single-stranded DNA binding (25). At such low substrate concentrations, greater than 90% of the enzyme population is not bound to the DNA at equilibrium.…”

“…In the present work, we also found the affinity of the S369P variant to be higher than that of the WT enzyme for both single-stranded DNA and double-stranded DNA (Table 2). Similarly, singlestranded DNA binding by the R374Q and W315L forms is reported to be both deficient (24,29) and proficient (13) by EMSA. Although the current work concurs with the near WT binding affinity previously observed for the A359T (13,24) and R334Q (29) variants, the weak DNA binding reported for the K319E (13), L381P and I367T (24), and P335L (29) variants is difficult to reconcile with the high affinity binding that we observed (Table 1).…”