Our long-term goal is to help decipher how cells process DNA lesions in the maintenance of genomic integrity. The DNA damage response (DDR) involves protein complexes that sense DNA lesions and coordinate cell cycle progression along with DNA repair, apoptosis and DNA damage tolerance pathways. Reversible post-translational modifications (PTMs) of histone and non-histone proteins are essential for the DDR machinery to assemble at DNA damage sites but the mechanisms are poorly understood. Our proposed research focuses on probing, from a structural perspective, how PTMs participate in the control of time- and space-dependent protein associations in the DDR. Specifically, we will investigate the roles of lysine methylation and ubiquitylation in the repair of DNA double-strand breaks (DSBs) and in DNA damage tolerance by translesion DNA synthesis (TLS). 53BP1 is an important mediator of DNA repair by non-homologous end joining. In response to DSBs, 53BP1 associates with histone H4 dimethylated at lysine 20 (H4K20me2), which, in the absence of damage, is thought to be complexed to the lysine demethylase JMJD2A. Upon DSB induction, several ubiquitin ligases such as RNF8 are recruited to damage sites where they ubiquitylate histones and JMJD2A. The ubiquitylation of JMJD2A triggers its degradation, facilitating 53BP1 interaction with H4K20me2 and relocalization to DSBs. Under Aim 1, we will probe the recruitment mechanisms of 53BP1, JMJD2A and structurally related protein PHF20. We will characterize interactions of these proteins with the nucleosome core particle. Under Aim 2, we will investigate how mono-ubiquitylation of the DNA clamp PCNA in response to DNA damage activates damage tolerance by TLS. We will probe how the TLS DNA polymerase Rev1 and TLS are activated by PCNA ubiquitylation. Our hypothesis is that ubiquitylation alters PCNA–Rev1 interaction and functions as a double-switch to turn on TLS. Our studies will be the basis for designing in vivo assays that can elevate our knowledge of the DDR. Alteration of H4K20 methylation is a hallmark of human tumor cells and mutations in several DDR proteins are linked to cancer predispositions in humans. Because loss of 53BP1 reverses cancer phenotypes of BRCA1 mutant cells, inhibition of 53BP1 recruitment to DSB sites could have potential for cancer therapy. Rev1 and TLS contribute to the acquired therapeutic resistance of cancer cells.