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Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers.


ABSTRACT: Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.

SUBMITTER: Craig JM 

PROVIDER: S-EPMC5692567 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers.

Craig Jonathan M JM   Laszlo Andrew H AH   Brinkerhoff Henry H   Derrington Ian M IM   Noakes Matthew T MT   Nova Ian C IC   Tickman Benjamin I BI   Doering Kenji K   de Leeuw Noah F NF   Gundlach Jens H JH  

Proceedings of the National Academy of Sciences of the United States of America 20171016 45


Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during tra  ...[more]

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