Enzymatic Activities Involved in the DNA Resynthesis Step of Nucleotide Excision Repair Are Firmly Attached to Chromatin

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Abstract
In this study the role of nuclear architecture in nucleotide excision repair (NER) was investigated by gentle dismantling of the cell and probing the capability of chromatin to carry out repair in vitro. The rationale behind this approach is that compartmentalization of NER at nuclear structures would make the enzymatic activities refractory to extraction by buffers that solubilize cellular membranes. In order to obtain intact chromatin primary human fibroblasts were encapsulated in agarose microbeads and lysed in isotonic buffers containing the non-ionic detergent Triton X-100. Under these conditions the majority of cellular proteins diffuse out of the beads, but the remaining chromatin is able to replicate and to transcribe DNA in the presence of triphosphates and Mg2+. UV irradiation of confluent repair-proficient human fibroblasts prior to lysis stimulated the incorporation of deoxynucleotide triphosphates in Triton X-100-isolated chromatin, even under stringent lysis conditions. In addition, experiments with UV-sensitive xeroderma pigmentosum (complementation groups A and C) and Cockayne's syndrome fibroblasts (complementation group A) revealed that this repair synthesis was due to global genome repair activity. Transcription-coupled repair was only detectable in cells permeabilized by streptolysin O (SLO). Repair synthesis in Triton X-100-isolated chromatin amounted to 15% of the total repair synthesis as measured in SLO-permeabilized cells. To allow the detection of these activities in vitro, presynthesis complexes have to be formed in intact cells, indicating that chromatin from Triton X-100-lysed cells is unable to initiate NER in vitro. Our data indicate that the components involved in the resynthesis step of NER are tightly associated with chromatin. A substantial fraction of total proliferating cell nuclear antigen (PCNA), which is required for the resynthesis step in NER, has been reported to become Triton X-100 non-extractable and tightly associated with nuclear structures after UV irradiation of cells. We propose that Triton X-100-resistant repair synthesis might be mediated by this chromatin-bound fraction of total PCNA.