To investigate how the nucleotide excision repair initiator XPC locates DNA damage in mammalian cell nuclei we analyzed the dynamics of GFP-tagged XPC. Photobleaching experiments showed that XPC constantly associates with and dissociates from chromatin in the absence of DNA damage. DNA-damaging agents retard the mobility of XPC, and UV damage has the most pronounced effect on the mobility of XPC-GFP. XPC exhibited a surprising distinct dynamic behavior and subnuclear distribution compared with other NER factors. Moreover, we uncovered a novel regulatory mechanism for XPC. Under unchallenged conditions, XPC is continuously exported from and imported into the nucleus, which is impeded when NER lesions are present. XPC is omnipresent in the nucleus, allowing a quick response to genotoxic stress. To avoid excessive DNA probing by the low specificity of the protein, the steady-state level in the nucleus is controlled by nucleus-cytoplasm shuttling, allowing temporally higher concentrations of XPC in the nucleus under genotoxic stress conditions.

The replication of the undamaged genomic DNA requires error-free DNA polymerases δ and ϵ as part of a protein complex that acts continuously along the double helix. In contrast, when the genomic structure is perturbed, DNA replication needs to function more flexibly to bypass DNA distortions. It has been proposed that the newly discovered error prone DNA polymerases play a role in the replication of irregular structure. Here we report that one of them, the human Polκ, is mostly localised uniformly in the nucleus of undamaged cells, but could be also concentrated in PCNA-containing replication foci. Following treatment with anti-replicative agents, the proportion of foci-containing cells was increased. These data suggest that Polκ may function as part of the replication machinery itself and could be recruited when replicative complexes are stalled. Mutagenesis experiments also indicated that Polκ involvement may affect the accuracy of DNA replication. The results are discussed within the context of the oncogenic process since Polκ has been found as overexpressed in some cancers.

No abstract