SUMMARY Human DNA polymerase delta (Pol δ) forms a holoenzyme complex with the DNA sliding clamp proliferating cell nuclear antigen (PCNA) to perform its essential roles in genome replication. Here, we utilize live-cell single-molecule tracking to monitor Pol δ holoenzyme interaction with the genome in real time. We find holoenzyme assembly and disassembly in vivo are highly dynamic and ordered. PCNA generally loads onto the genome before Pol δ. Once assembled, the holoenzyme has a relatively short lifetime on the genome, implying multiple Pol δ binding events may be needed to synthesize an Okazaki fragment. During disassembly, Pol δ dissociation generally precedes PCNA unloading. We also find that Pol δ p125, the catalytic subunit of the holoenzyme, is maintained at a constant cellular level, indicating an active mechanism for control of Pol δ levels in vivo . Collectively, our studies reveal that Pol δ holoenzyme assembly and disassembly follow a predominant pathway in vivo ; however, alternate pathways are observed.

Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair.

Ciona intestinalis, a common sea squirt, exhibits lower reproductive success at the upper extreme of the water temperatures it experiences in coastal New England. In order to understand the changes in protein expression associated with elevated temperatures, and possible response to global temperature change, we reared C. intestinalis from embryos to adults at 18°C (a temperature at which they reproduce normally at our collection site in Rhode Island) and 22°C (the upper end of the local temperature range). We then dissected ovaries from animals at each temperature, extracted protein, and measured proteomic levels using shotgun mass spectrometry (LC-MS/MS). 1532 proteins were detected at a 1% false discovery rate present in both temperature groups by our LC-MS/MS method. 62 of those proteins are considered up- or down-regulated according to our statistical criteria. Principal component analysis shows a clear distinction in protein expression pattern between the control (18°C) group and high temperature (22°C) group. Similar to previous studies, cytoskeletal and chaperone proteins are upregulated in the high temperature group. Unexpectedly, we find evidence that proteolysis is downregulated at the higher temperature. We propose a working model for the high temperature response in C. intestinalis ovaries whereby increased temperature induces upregulation of signal transduction pathways involving PTPN11 and CrkL, and activating coordinated changes in the proteome especially in large lipid transport proteins, cellular stress responses, cytoskeleton, and downregulation of energy metabolism.

Fanconi anemia (FA) is a human genetic disease characterized by congenital defects, bone marrow failure, and increased cancer risk. FA is associated with mutation in one of 24 genes. The protein products of these genes function cooperatively in the FA pathway to orchestrate the repair of DNA interstrand cross-links. Few model organisms exist for the study of FA. Seeking a model organism with a simpler version of the FA pathway, we searched the genome of the simple chordate Ciona intestinalis for homologs of the human FA-associated proteins. BLAST searches, sequence alignments, hydropathy comparisons, maximum likelihood phylogenetic analysis, and structural modeling were used to infer the likelihood of homology between C. intestinalis and human FA proteins. Our analysis indicates that C. intestinalis indeed has a simpler and potentially functional FA pathway. The C. intestinalis genome was searched for candidates for homology to 24 human FA and FA-associated proteins. Support was found for the existence of homologs for 13 of these 24 human genes in C. intestinalis. Members of each of the three commonly recognized FA gene functional groups were found. In group I, we identified homologs of FANCE, FANCL, FANCM, and UBE2T/FANCT. Both members of group II, FANCD2 and FANCI, have homologs in C. intestinalis. In group III, we found evidence for homologs of FANCJ, FANCO, FANCQ/ERCC4, FANCR/RAD51, and FANCS/BRCA1, as well as the FA-associated proteins ERCC1 and FAN1. Evidence was very weak for the existence of homologs in C. intestinalis for any other recognized FA genes. This work supports the notion that C. intestinalis, as a close relative of vertebrates, but having a much reduced complement of FA genes, offers a means of studying the function of certain FA proteins in a simpler pathway than that of vertebrate cells.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci. The regulation and function of FANCD2 and FANCI, however, is poorly understood. In addition, how chromatin state impacts pathway activation is also unknown. In this study, we have examined the influence of chromatin state on the activation of the FA pathway. We describe potent activation of FANCD2 and FANCI monoubiquitination and nuclear foci formation following treatment of cells with the histone methyltransferase inhibitor BRD4770. BRD4770-induced activation of the pathway does not occur via the direct induction of DNA damage or via the inhibition of the G9a histone methyltransferase, a mechanism previously proposed for this molecule. Instead, we show that BRD4770-inducible FANCD2 and FANCI monoubiquitination and nuclear foci formation may be a consequence of inhibition of the PRC2/EZH2 chromatin-modifying complex. In addition, we show that inhibition of the class I and II histone deacetylases leads to attenuated FANCD2 and FANCI monoubiquitination and nuclear foci formation. Our studies establish that chromatin state is a major determinant of the activation of the FA pathway and suggest an important role for the PRC2/EZH2 complex in the regulation of this critical tumor suppressor pathway.