Mutation of a predicted DNA binding site on bacteriophage T4 Rad50 creates a constitutively active ATPase

Abstract

The Mre11/Rad50 (MR) complex uses ATP to repair DNA double strand breaks. The mechanistic details of how ATP hydrolysis relates to DNA binding and processing, as well as the associated structural changes that occur within the MR complex heterotetramer are not fully understood. An amino acid sequence‐based analysis predicted a patch of positively charged residues on bacteriophage T4 Rad50 as a potential DNA binding site. Specific mutations were generated to neutralize the charge at this site and the mutant proteins were biochemically characterized. Consistent with the bioinformatic predictions, the mutant proteins bound double‐stranded DNA with lesser affinity than the wild‐type enzyme. Surprisingly, ATP hydrolysis assays indicated that the mutations created an enzyme that was fully activated, even in the absence of bound activator DNA. DNA exonuclease assays suggest that the ATP hydrolyzed by the mutant proteins is somehow uncoupled from normal repetitive cleavage of nucleotides by the MR complex. All together, these findings suggest that the point mutations have stabilized a conformational state of Rad50 that is normally induced by DNA binding and clearly demonstrate the allosteric relationship between the substrate DNA and the activator ATP. A complete investigation of these residues and their effect on allostery will lead to a more complete picture of the steps involved in the repair of harmful DNA breaks.Support or Funding InformationNSF. Award number 1716269. The molecular mechanisms of the Mre11/Rad50 DNA repair complexThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.