Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required.

Complete activation of signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Y701 and a conserved PMS 727 P sequence. S727 phosphorylation of STAT1 in interferon-g (IFN-g)-treated mouse ®broblasts occurred without a need for p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 or c-Jun kinases, and required both an intact SH2 domain and phosphorylation of Y701. In contrast, UV irradiation-induced STAT1 phosphorylation on S727 required p38MAPK, but no SH2 domain± phospho-tyrosine interactions. Mutation of S727 differentially affected IFN-g target genes, at the level of both basal and induced expression. Particularly strong effects were noted for the GBP1 and TAP1 genes. The PMS 727 P motif of STAT3 was phosphorylated by stimuli and signaling pathways different from those for STAT1 S727. Transfer of the STAT3 C-terminus to STAT1 changed the stimulus and pathway speci®city of STAT1 S727 phosphorylation to that of STAT3. Our data suggest that STAT C-termini contribute to the speci®city of cellular responses by linking individual STATs to different serine kinase pathways and through an intrinsically different requirement for serine phosphorylation at different target gene promoters.

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) and are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that pol is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of pol in foci, it results in an increased residence time in foci. pol is even more mobile than pol, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both pol and pol diffuse through the cell but that they are transiently immobilized for ϳ150 ms, with a larger proportion of pol than pol immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of pol but not pol. Our data are consistent with a model in which the polymerases are transiently probing the DNA/ chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.