ENZYMATIC-ACTIVITIES INVOLVED IN THE DNA RESYNTHESIS STEP OF NUCLEOTIDE EXCISION-REPAIR ARE FIRMLY ATTACHED TO CHROMATIN

In this study the role of nuclear architecture in nucleotide excision repair (NER) was investigated by gentle dismantling of the cell and pr obing the capability of chromatin to carry out repair in vitro. The ra tionale behind this approach is that compartmentalization of NER at nu clear structures would make the enzymatic activities refractory to ext raction by buffers that solubilize cellular membranes, In order to obt ain intact chromatin primary human fibroblasts were encapsulated in ag arose microbeads and lysed in isotonic buffers containing the non-ioni c detergent Triton X-100. Under these conditions the majority of cellu lar proteins diffuse out of the beads, but the remaining chromatin is able to replicate and to transcribe DNA in the presence of triphosphat es and Mg2+. UV irradiation of confluent repair-proficient human fibro blasts 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 xeroderm a pigmentosum (complementation groups A and C) and Cockayne's syndrome fibroblasts (complementation group A) revealed that this repair synth esis was due to global genome repair activity. Transcription-coupled r epair was only detectable in cells permeabilized by streptolysin O (SL O), 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 co mplexes have to be formed in intact cells, indicating that chromatin f rom Triton X-100-lysed cells is unable to initiate NER in vitro, Our d ata indicate that the components involved in the resynthesis step of N ER are tightly associated with chromatin, A substantial fraction of to tal proliferating cell nuclear antigen (PCNA), which is required for t he resynthesis step in NER, has been reported to become Triton X-100 n on-extractable and tightly associated with nuclear structures after UV irradiation of cells, We propose that Triton X-100-resistant repair s ynthesis might be mediated by this chromatin-bound fraction of total P CNA.