title: DNA sequences proximal to human mitochondrial DNA deletion breakpoints prevalent in human disease form G-quadruplexes, a class of DNA structures inefficiently unwound by the mitochondrial replicative Twinkle helicase.
abstract: Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the "Pattern Finder" G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.
authors.affiliation: From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, NIH Biomedical Research Center, Baltimore, Maryland 21224.
authors.forename: Sanjay Kumar
authors.initials: SK
authors.lastname: Bharti
authors.affiliation: From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, NIH Biomedical Research Center, Baltimore, Maryland 21224.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Sommers
authors.affiliation: the ARNA Laboratory, University of Bordeaux, F-33000 Bordeaux, France, INSERM U869, Institut Européen de Chimie et Biologie (IECB), F-33600 Pessac, France.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.affiliation: the Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida 32312.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Kaplan
authors.affiliation: the FinMIT Centre of Excellence, BioMediTech and Tampere University Hospital, Pirkanmaa Hospital District, University of Tampere, FI-33014 Tampere, Finland, and the Department of Pediatrics, Nijmegan Centre for Mitochondrial Disorders, Radboud University Medical Centre, Geert Grooteplein 10, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors.forename: Johannes N
authors.initials: JN
authors.lastname: Spelbrink
authors.affiliation: the ARNA Laboratory, University of Bordeaux, F-33000 Bordeaux, France, INSERM U869, Institut Européen de Chimie et Biologie (IECB), F-33600 Pessac, France.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Mergny
authors.affiliation: From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, NIH Biomedical Research Center, Baltimore, Maryland 21224, broshr@mail.nih.gov.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Brosh
mesh_terms: D000375:Aging / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D019295:Computational Biology; D017124:Conserved Sequence / Q000235:genetics; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics*; D004194:Disease / Q000235:genetics*; D019143:Evolution, Molecular; D054856:G-Quadruplexes; D054629:Genome, Mitochondrial / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics; D009691:Nucleic Acid Denaturation; D059372:Nucleotide Motifs / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*; D013379:Substrate Specificity; D016615:Telomere / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D004265:DNA Helicases; C431576:TWNK protein, human
keywords: DNA Helicase; DNA Replication; G-quadruplex; Genetic Disease; Genomic Instability; Mitochondria; Twinkle