
CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism
Author(s) -
Nina Y. Yao,
Dan Zhang,
Olga Yurieva,
Mike O'Donnell
Publication year - 2022
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.2119580119
Subject(s) - helicase , atp hydrolysis , adenosine triphosphate , dna , nucleotide , biophysics , walker motifs , atp synthase , limiting , chemistry , biochemistry , enzyme , biology , atpase , rna , gene , mechanical engineering , engineering
Significance The rate-limiting step of hexameric replicative helicases is ill-understood and this study on the eukaryotic replicative helicase CMG identifies that the catalytic ATP hydrolysis step is not the rate-limiting step, implying that a conformational change may be rate-limiting for DNA-unwinding activity. The results support a “staircase” model of CMG movement along DNA, requiring a large conformational change after ATP hydrolysis. Many studies use ATPγS to “preload” CMG onto DNA. However, this study demonstrates that ATPγS hydrolysis fuels CMG to unwind DNA. To achieve preloading without unwinding, the current study provides an alternative nucleotide analog, AMP-PNP, that is not hydrolyzable and efficiently preloads CMG onto DNA.