53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions

The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by non-homologous end joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and anti-recombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double strand breaks (DSBs). Here we show that a 53BP1 phospho-mutant 53BP1(8A), comprising alanine substitutions of the 8 most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1 deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP1(8A) recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP1(8A). We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phospho-dependent interactions with RIF1 and PTIP

In vivo resolution of oligomers with fluorescence photobleaching recovery histograms

Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity

Nup98 is a mobile nucleoporin with transcription-dependent dynamics

Nucleoporin 98 (Nup98), a glycine-leucine-phenylalanine-glycine (GLFG) amino acid repeat-containing nucleoporin, plays a critical part in nuclear trafficking. Injection of antibodies to Nup98 into the nucleus blocks the export of most RNAs. Nup98 contains binding sites for several transport factors; however, the mechanism by which this nucleoporin functions has remained unclear. Multiple subcellular localizations have been suggested for Nup98. Here we show that Nup98 is indeed found both at the nuclear pore complex and within the nucleus. Inside the nucleus, Nup98 associates with a novel nuclear structure that we term the GLFG body because the GLFG domain of Nup98 is required for targeting to this structure. Photobleaching of green fluorescent protein-Nup98 in living cells reveals that Nup98 is mobile and moves between these different localizations. The rate of recovery after photobleaching indicates that Nup98 interacts with other, less mobile, components in the nucleoplasm. Strikingly, given the previous link to nuclear export, the mobility of Nup98 within the nucleus and at the pore is dependent on ongoing transcription by RNA polymerases I and II. These data give rise to a model in which Nup98 aids in direction of RNAs to the nuclear pore and provide the first potential mechanism for the role of a mobile nucleoporin