M.Sc. Graduate Seminar-Roi Siegelman
25/11/2020 13:00

A single molecule study of theroles of the RecC subunit in DNA unwinding by RecBCD

 DNAdouble-strand breaks (DSBs) are the most cytotoxic of DNA lesions and theirrepair requires a comprehensive and rapid cellular response. In EscherichiaColi,RecBCDinitiates the homologous recombination pathway for DSB repair by unwinding theDNA at the damaged site rapidly and processively. Asingle-molecule assay previously developed in our lab, enabled monitoring theactivity of the individual subunits, as part of the full complex of RecBCD. Theresults demonstrated that synergy between its subunits supports unwinding by RecBCD.However, how RecCcontributes to RecBCD’sunwinding activity has not yet been fully understood. In my work, I combinedsingle-molecule and in vivoassays to probe the activity of RecBCD mutated in their “pin” domain within RecC,which was suggested to split the duplex DNA before the individual strands arepulled by RecB and RecD.  Our single molecule results reveal that,while the velocity is not significantly affected by these mutations, the pindomain is crucial for the processivity of the whole complex and its subunits.Moreover, the in vivoexperiments suggest that the processivity determines the ability of RecBCD toefficiently initiate the repair reaction.