期刊名称:Proceedings of the National Academy of Sciences
印刷版ISSN:0027-8424
电子版ISSN:1091-6490
出版年度:2022
卷号:119
期号:4
DOI:10.1073/pnas.2119580119
语种:English
出版社:The National Academy of Sciences of the United States of America
摘要: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.
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.