CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism.
Proc Natl Acad Sci U S A
; 119(4)2022 01 25.
Article
em En
| MEDLINE
| ID: mdl-35042821
ABSTRACT
The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding.
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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Trifosfato de Adenosina
/
DNA Helicases
/
Complexos Multiproteicos
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2022
Tipo de documento:
Article