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oai:figshare.com:article/34035564 2026-09-30T17:35:02Z category_4 category_12 category_13 category_14 category_734 portal_5 item_type_6 month_year_09_2026

<p>List of plasmids used in this study.</p> Luis E. Martínez-Rodríguez (25145346) Stephen P. Bell (25145349) Biochemistry Cell Biology Genetics Molecular Biology Biological Sciences not elsewhere classified origin recognition complex saccharomyces cerevisiae </ helicase loading activity div >< p atpase interfaces within six orc subunits orc atp hydrolysis defective orc allele atp hydrolysis orc subunits defective orc vitro </ toa2 </ r267a </ sixth allele reducing transcription orc4r ). lethal phenotype genetic screen essential role cdc6 complex binds origins although functions <div><p>The origin recognition complex (ORC) binds origins of replication and directs the loading of the Mcm2–7 replicative helicase at these sites. Five of the six ORC subunits are related to the AAA+ family of ATPases. Although functions for ATP hydrolysis by Cdc6 and the Mcm2–7 complex have been described, the essential role of ORC ATP hydrolysis remains unclear. We performed a genetic screen in <i>Saccharomyces cerevisiae</i> for suppressors of the lethal phenotype of the <i>orc4-R267A</i> allele, which disrupts ORC ATP hydrolysis <i>in vitro</i>. We identified six causative mutations, five of which are distributed across different ORC subunits. The suppressor mutations in Orc1 and Orc4, but not those in other ORC subunits, increase the <i>in vitro</i> helicase loading activity of ATPase-defective ORC (ORC4R). Allele specificity studies show the alleles specifically suppress defects at ATPase interfaces within the ORC-Cdc6 complex. The sixth allele is a mutation in <i>TOA2</i>, a subunit of the TFIIA general transcription factor. Mutations in the general transcription factors TBP and TFIIB, and the large subunit of RNA Polymerase II also suppress <i>orc4-R267A</i> lethality, suggesting that reducing transcription is sufficient for suppression<i>.</i> Our study identifies multiple pathways to suppress the lethal phenotype of an ATPase-defective ORC allele and reveals a connection between ORC ATP hydrolysis and transcription.</p></div> 2026-09-30T17:34:54Z Text Journal contribution 10.1371/journal.pone.0349465.s008 https://figshare.com/articles/journal_contribution/_p_List_of_plasmids_used_in_this_study_p_/34035564 CC BY 4.0