Nucleic Acids Research, Vol 25, Issue 5 1056-1063, Copyright © 1997 by Oxford University Press
K Bouayadi, A van der Leer-van Hoffen, AS Balajee, AT Natarajan, AA van Zeeland and LH Mullenders
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.
ARTICLES
Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin
MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, Leiden, The Netherlands.
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