Understanding the Cell‐Cycle Dependence of Mus81‐Mms4 Recruitment and its Reliance on Slx4

DNA damage repair pathways are a key element in maintaining genomic stability and preventing cancer and other diseases, particularly given the prevalence of DNA damaging agents. One form of DNA damage is double‐strand breaks (DSBs), the repair of which utilizes numerous repair proteins regardless of which subpathway is implemented. One such repair protein in S. cerevisiae is Mus81, which forms a complex with Mms4, that functions as a structure‐specific endonuclease that cleaves flapped DNA structures present in intertwined chromosomes that have undergone DSB repair in late S and G2 phases. Here, we seek to understand the cell cycle dependence of Mus81‐ recruitment to DSBs and determine if this recruitment is Slx4‐dependent. This exploration requires complete cell cycle synchronization with □‐factor followed by release back into cell cycle following □‐factor removal. As they progress through the cell cycle, cultures are monitored by fluorescence microscopy and flow cytometry to properly correlate fluorescence data with discrete stages of the cell cycle. The yeast strain under study contains a Mus81 protein tagged with yEmRFP which enables its tracking during the fluorescence microscopy portion of the experiment. Preliminary microscopy experiments on a Mus81‐yEmRFP labeled strain have demonstrated that Mus81 is present in low abundance, making it difficult to track the RFP signal throughout the cell cycle. This creates the need for increased expression of Mus81 if it is to be investigated via fluorescence microscopy. Therefore, a plasmid has been constructed that incorporates an ADH1 promoter in front of the Mus81‐yEmRFP gene to enable overexpression. The use of this modified strain will allow for experimental correlation of Mus81‐Mms4 activity with a specific stage of the cell cycle. Support or Funding Information National Institutes of Health (NIH) (Grant SC1GM127204)NIH BUILD Program (NIH Grant 5TL4GM118977)