Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase

► Processivity of DNA glycosylases can cause double-strand breaks at clustered lesions. ► We engineered pyrimidine dimer-specific DNA glycosylases with reduced processivity. ► We developed an assay to quantitatively measure incisions at clustered abasic sites. ► Biochemical and computational data reveal residues critical for processive nicking. ► Enzymes with reduced processivity but full catalytic function can be engineered.