Xeroderma pigmentosum endonuclease complexes show reduced activity on and affinity for psoralen cross-linked nucleosomal DNA.
Two DNA endonuclease complexes have been isolated from the chromatin of normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells which are active on DNA damaged with psoralen plus long wavelength ultraviolet radiation (UVA). In both normal and XPA cells, one endonuclease complex, pI 4.6, recognizes the psoralen cross-link and the other endonuclease complex, pI 7.6, recognizes the psoralen monoadduct. The levels of activity of these complexes from both normal and XPA cells are similar on damaged naked DNA. Kinetic analysis of assays using graduated concentrations of substrate revealed that selective activity of these endonuclease complexes on 8-MOP plus UVA treated DNA correlates with a reduction in Km of these complexes, indicating an increased affinity for, or rate of association with, damaged naked DNA. When the damaged substrates were reconstituted into core nucleosomes (without histone H1), both normal endonuclease complexes showed a 2.5-fold enhancement of activity, which correlated kinetically with a further increase in affinity, or rate of association (decreased Km), for this damaged nucleosomal substrate. This increase in activity and in affinity was reduced but not eliminated when histone H1 was present. By contrast, neither XPA endonuclease complex showed this enhanced activity on, or affinity for, damaged core nucleosomal DNA, and actually showed decreased activity, and affinity, when histone H1 was present. Introduction, via electroporation, of either of the normal complexes into 8-MOP plus UVA treated XPA cells in culture corrected their DNA-repair defect, further confirming the role of these complexes in the repair process.
Parrish
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DD
Department of Laboratory Medicine and Pathology, UMDNJ, New Jersey Medical School, Newark 07103-2757.
Lambert
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WC
Lambert
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MW
D002461:Cell Line, Transformed; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005363:Ficusin / Q000494:pharmacology*; D005816:Genetic Complementation Test; D006657:Histones / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D009707:Nucleosomes / Q000378:metabolism; D010957:Plasmids; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000201:enzymology*
D006657:Histones; D009707:Nucleosomes; D004247:DNA; D003850:Deoxyribonuclease I; D005363:Ficusin