Edited by James E. Haber, Brandeis University, Waltham, MA, and approved February 4, 2021 (received for review October 29, 2020)DNA resection is an initial, decisive step in the homologous recombination repair pathway of DNA double-strand breaks (DSBs). However, the individual roles and cross-talk of key proteins in this process remain unclear. To resolve the spatiotemporal dynamics of this intricate process, we applied multicolor single-molecule localization microscopy along with robust analytical approaches. Our data reveal the dynamic actions and interactions of MRE11, BRCA1, and CtIP...…Edited by James E. Haber, Brandeis University, Waltham, MA, and approved February 4, 2021 (received for review October 29, 2020)DNA resection is an initial, decisive step in the homologous recombination repair pathway of DNA double-strand breaks (DSBs). However, the individual roles and cross-talk of key proteins in this process remain unclear. To resolve the spatiotemporal dynamics of this intricate process, we applied multicolor single-molecule localization microscopy along with robust analytical approaches. Our data reveal the dynamic actions and interactions of MRE11, BRCA1, and CtIP...WW…