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A helicase is born.
Author: Carlos T. Moraes
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"news & views 200 nature genetics ? volume 28 ? july 2001 For more than a decade, neuromuscular disease experts have been puzzled by a clinical syndrome characterized by the lack of eye movements?a condition known as progressive external ophthalmoplegia (PEO). In spo- radic cases, each individual car- ries one deletion, which is found in the majority of her/his skeletal muscle mitochondrial genomes 1 . In most inherited cases, however, the muscle mitochondrial DNA (mtDNA) of each individual is damaged by many and diverse deletions 2 . This inherited disor- der provided the first evidence that mutation of a nuclear gene can affect the integrity of mtDNA. On page 223 of this issue, Johannes Spelbrink and colleagues 3 provide a molecular clue about the nuclear-mito- chondrial interactions underly- ing this disorder. Autosomal dominant inheritance of the disease is much more frequent than its recessive forms, and has been associated with three different loci in different fami- lies: 10q24, 3p14?21 and 4q34?35. The gene on chromosome 4q encodes adenine nucleotide translocator 1 (ANT1), a protein that controls ATP and ADP shuttling at the mitochondrial inner membrane in muscle cells 4 . The mechanism by which mutation of ANT1 affects mtDNA integrity is unknown, but it may involve the stalling of the mitochondrial DNA polymerase ?, due to an imbalance in the mitochondrial deoxynucleotide pool. Spelbrink and colleagues 3 have now identified the gene mutated in the 10q-linked adPEO fami- lies?C10orf2. This gene encodes a protein with homology to a T7 bacteriophage helicase/primase, the bifunctional enzyme encoded by the T7 gene 4. The homology, however, is significant A helicase is born Carlos T. Moraes Departments of Neurology and Cell Biology & Anatomy, University of Miami School of Medicine. Miami, Florida 33136, USA. e-mail: cmoraes@med.miami.edu One of three loci previously associated with autosomal dominant progressive external ophthalmoplegia (adPEO) encodes ANT1, a mitochondrial nucleotide transporter. Now, mutations in two other genes are found in people with adPEO. One of these encodes a new helicase, Twinkle, which is related to the product of bacteriophage T7 gene 4, and co-localizes with mitochondrial DNA. The identification of Twinkle adds a new star to the expanding constellation of ?helicase diseases?. Mitochondrial star gazing. Twinkle is a novel helicase-like mitochondrial protein that co-localizes with the mitochondrial genome (immunostaining of bromodeoxyuridine-labeled cells; arrows in left panel). The mitochondr- ial marker in the right panel shows the organelle distribution. On page 223 of this issue, Spelbrink et al 3 . provide evidence that mutations in the gene coding for Twinkle are associated with multiple mitochondrial DNA dele- tions, and a neuromuscular disease, adPEO. Micrograph by Corina van Wav- eren (University of Miami). Mitchell et al., however, shows that genes that mediate differentiation can be trapped. The authors attribute their success to a potent drug-resistance marker that allows selection of genes with very low levels of transcription in ES cells. Choose your mutagen So how does gene trapping compare with chemical mutagenesis? The phenotype-dri- ven screens rely on a phenotype, and the majority of the lines with no detectable phe- notype obtained by gene trap would proba- bly not be found after chemical mutagenesis. Thus, careful analysis of gene-trap lines might help the phenotypists to develop bet- ter screens. One disadvantage of gene trap- ping is that, in general, the only allele generated is a null allele. In contrast, chemi- cal mutagenesis may produce partial loss-of- function mutations, and these can be useful in dissecting the different functions of a gene and its alternative splice variants. For exam- ple, an animal with a hypomorphic muta- tion may escape early lethality associated with a null allele, thus disclosing an affected process that occurs later in development. On the other hand, chemical mutagene- sis is stymied by the process of having to positionally clone the interesting muta- tions. The resolution of the mapping required, and how much needs to be done to reduce the candidate genes to a suffi- ciently small number for close examina- tion to find the causative mutation, is a hot topic of much debate among aficiona- dos. Both sides are signatories to the treaty establishing the International Mouse Mutagenesis Consortium 13 , with the com- mon goal of establishing a role for every mammalian gene. This challenge is of such a scale that every approach will be needed to tackle it. L50132 1. International Human Genome Sequencing Consortium. Nature 409, 860?921 (2001). 2. Venter J.C. et al. Science 291, 1304?1351 (2001). 3. Nolan, P.M. et al. Nature Genet. 25, 440?443 (2000). 4. De Angelis M.H. et al. Nature Genet. 25, 444?447 (2000). 5. Mitchell, K.J. et al. Nature Genet. 28,241?249 (2001). 6. Gossler, A. et al. Science 244, 463?465 (1989). 7. Freidrich, G. & Soriano, P. Genes Dev. 5, 1515?1523 (1991). 8. Von Melchmer, H. et al. Genes Dev. 6, 919?927 (1992). 9. Hicks, G.G. et al. Nature Genet. 16, 338?344 (1997). 10. Zambrowicz, B.P. et al. Nature 392, 608?611 (1998). 11. Wiles, M.V. et al. Nature Genet. 24, 13?14 (2000). 12. Skarnes, W.C. et al. Proc.Natl. Acad. Sci. U.S.A. 92, 6592?6596 (1995). 13. International Mouse Mutagenesis Consortium. Science 291, 1251?1255 (2001). � 2001 Nature Pub lishing Gr oup http://g enetics.nature . com � 2001 Nature Publishing Group http://genetics.nature.com news & views nature genetics ? volume 28 ? july 2001 201 only in the helicase domain. Helicases are enzymes that can unwind duplex DNA or RNA molecules; DNA helicases mediate DNA replication, repair, recombination, and transcription 5 , whereas RNA helicases are involved in transcription, RNA pro- cessing, regulation of RNA stability, ribo- some assembly, and translation 6 . The C10ORF2 gene product belongs to a class of hexameric helicases 7 , and its amino acid sequence is well conserved among multi- cellular eukaryotes. DNA or RNA helicases exist in bovine and human mitochondria, but their function in mammals is poorly understood 8,9 . Two helicases have been characterized in the mitochondria of Sac- charomyces cerevisiae, PIF1 and Hmi1p, neither of which has significant homology to the C10orf2 product. Interestingly, Hmi1p is necessary for the maintenance of intact, but not partially deleted, mtDNA 10 . The C10orf2 gene product co-localizes with mtDNA, giving rise to a punctate, star-like staining (hence Twinkle; see fig- ure). Twinkle also co-localizes with mito- chondrial nucleoids?ovoid bodies of about 0.3?0.6 �m in diameter that are well characterized in S. cerevisiae; where each nucleoid contains 3?4 copies of mtDNA and as many as 20 different polypeptides 11 . The association between C10orf2 mutations and multiple mtDNA dele- tions is an enigma. As mtDNA deletions accumulate during aging of normal indi- viduals, the effect of mutations in C10orf2 may be associated with acceler- ated mitochondrial aging. This parallels the phenotypes of mutations in genes encoding some nuclear helicases of the RecQ group: Bloom syndrome 12 (char- acterized by immunodeficiency, impaired fertility, dwarfism and predis- position to cancer), Werner syndrome 13 (affected people age prematurely with graying and thinning of the hair, and are susceptible to developing cataracts, type 2 diabetes mellitus, osteoporosis, athero- sclerosis and cancer) and some cases of Rothmund?Thomson syndrome 14 (which involves growth deficiency, pho- tosensitivity of the skin, cataracts, early graying and loss of hair, and increased susceptibility to cancer). The precise function of these different nuclear heli- cases remains to be determined, but their mutant versions are linked, at the cellu- lar level, by their common effects: increased genomic instability, including chromosomal breaks, multiple large deletions, and translocations 5 . If Twinkle proves to be a bona fide helicase, adPEO would be an additional helicase disorder associated with genomic instability and, as far as mitochondria are concerned, with premature aging. On dominance and deletions How could a dominant mutation in a heli- case lead to increased mtDNA deletions? Spelbrink and colleagues 3 propose two potential mechanisms. Because Twinkle may be assembled as a hexamer (like its homologue, the T7 gene 4 product), muta- tions affecting subunit interactions could lead to a dominant-negative effect. They could not, however, detect a defect in the oligomerization of adPEO-modified Twin- kle in cultured cells. Alternatively, muta- tions could lead to increased nucleotide hydrolysis, potentially altering the deoxynu- cleoside phosphate pool inside mitochon- dria. Indirect support for such a mechanism comes from the association with multiple mtDNA deletions of two genes involved in either the metabolism or the transport of nucleoside phosphates: ANT1, the adenine nucleotide transporter, and thymidine phosphorylase, a component of the thymi- dine salvage pathway, which is mutated in people with a disorder characterized by gas- trointestinal symptoms 15 . It is therefore possible that an imbal- ance in the mitochondrial nucleotide pool is a common feature of diseases associated with multiple mtDNA deletions. Alterna- tively, multimeric Twinkle complexes con- taining defective subunits could be less processive when encountering different forms of DNA damage 16 . If Twinkle par- ticipates in the replication of mtDNA, these relatively rare events could lead to the stalling of polymerase ? and an increase in the recombination rate. A link between defects in DNA poly- merase ? activity and multiple mtDNA dele- tions is strengthened by the report of Gert Van Goethem and colleagues 17 on page 213. They have found that mutations in the DNA polymerase ? are associated with both adPEO and an autosomal recessive variant of the disease. Taken together, these reports confirm earlier suspicions that the mtDNA replication/repair machinery is involved in at least some cases of PEO with mtDNA deletions inherited as a Mendelian trait 2 . The next step will be to demonstrate that Twinkle is indeed a helicase in vivo. Spel- brink and colleages 3 show that overexpres- sion of a C10orf2 cDNA in cultured human cells results in a very modest increase in helicase activity on artificial DNA/DNA templates in vitro. The possibility remains that Twinkle is an RNA helicase, or that it might use its helicase domain simply to bind DNA. Irrespective of its physiological function, the discovery of Twinkle and its mutant in adPEO is a significant step towards understanding the complex group of disorders involving nuclear-mitochon- drial miscommunication. L50132 1. Moraes, C.T. et al. N. Engl. J. Med. 320, 1293?1299 (1989). 2. Zeviani, M. et al. Nature 339, 309?311 (1989). 3. Spelbrink, J.N. et al. Nature Genet. 28, 223?231 (2001). 4. Kaukonen, J. et al. Science 289, 782?785 (2000). 5. Karow, J.K., Wu, L. & Hickson, I.D. Curr. Opin. Genet. Dev. 10, 32?38 (2000). 6. Luking, A., Stahl, U. & Schmidt, U. Crit. Rev. Biochem. Mol. Biol. 33, 259?296 (1998). 7. Patel, S.S. & Picha, K.M. Annu. Rev. Biochem. 69, 651?697 (2000). 8. Hehman, G.L. & Hauswirth, W.W. Proc. Natl. Acad. Sci. USA 89, 8562?8566 (1992). 9. Valgardsdottir, R., Brede, G., Eide, L.G., Frengen, E. & Prydz, H.P. J. Biol. Chem (in press). 10. Sedman, T., Kuusk, S., Kivi, S. & Sedman, J. Mol. Cell. Biol. 20, 1816?1824 (2000). 11. Lecrenier, N. & Foury, F. Gene 246, 37-48 (2000). 12. Ellis, N.A. et al. Cell 83, 655?666 (1995). 13. Yu, C.E. et al. Science 272, 258?262 (1996). 14. Kitao, S. et al. Nature Genet. 22, 82?84 (1999). 15. Nishino, I., Spinazzola, A. & Hirano, M. Science 283, 689?692 (1999). 16. Villani, G. & Tanguy Le Gac, N. J. Biol. Chem. 275, 33185?33188 (2000). 17. Van Goethem, G., L�fgren, A., Dermaut, B. Martin, J.J. & Van Broeckhoven, C. Nature Genet. 26, 213?214 (2001). � 2001 Nature Pub lishing Gr oup http://g enetics.nature . com � 2001 Nature Publishing Group http://genetics.nature.com "
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