ArticleThe Journal of biological chemistry2011
A257T linker region mutant of T7 helicase-primase protein is defective in DNA loading and rescued by T7 DNA polymerase.
Article in The Journal of biological chemistry, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 18 citations in OpenAlex.
- Structural insight and characterization of human Twinkle helicase in mitochondrial disease.Proceedings of the National Academy of Sciences of the United States of America · 2022Article
- DNA replication machinery: Insights fromComputational and structural biotechnology journal · 2021Review
- The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication.BMC molecular biology · 2018Article
- The Polyphyletic Origins of Primase-Helicase Bifunctional Proteins.Journal of molecular evolution · 2017Article
- DNA looping mediates nucleosome transfer.Nature communications · 2016Article
- Biocompatible and High Stiffness Nanophotonic Trap Array for Precise and Versatile Manipulation.Nano letters · 2016Article
- Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.Nucleic acids research · 2016Article
- Structure, function and evolution of the animal mitochondrial replicative DNA helicase.Critical reviews in biochemistry and molecular biology · 2016Review
- On human disease-causing amino acid variants: statistical study of sequence and structural patterns.Human mutation · 2015Article
- Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase-helicase from Arabidopsis thaliana.Molecular biology reports · 2014Article
- Helicase and polymerase move together close to the fork junction and copy DNA in one-nucleotide steps.Cell reports · 2014Article
- Loading mechanisms of ring helicases at replication origins.Molecular microbiology · 2012Review
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Authors and funding
8 authors at 3 institutions in 1 country.
Funding
Abstract
The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ∼75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.
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