Kinetic analysis of UV-induced incision discriminates between fibroblasts from different xeroderma pigmentosum complementation groups, XPA heterozygotes and normal individuals

Abstract

The capacity of a variety of human fibroblasts to incise DNA following exposure to far ultraviolet-light is determined from the rate of single-strand DNA break accumulation in the presence of DNA synthesis inhibitors. We have quantitated incision, one of the early steps in the UV excision repair pathway, in cells form normal, xeroderma pigmentosum groups C, D, G, H and variant individuals, and in the parents of one XPA patient. On the basis of the estimated initial rates of incision the different XP cells examined in this work can be marked as follows: XP variant ⋙ XPH > XPD > XPC > XPG > XPA . In each cell strain breaks accumulate immediately after irradiation over a range of 0.5–20 Jm −2 with the exception of the XPC strain examined, where there is an initial delay of 15 min. The rate of incision in XPA heterozygote cells is roughly half that of normal fibroblasts. Analysis of the kinetics of break accumulation over short intervals after irradiation permits estimation of the apparent enzymatic parameters, K m and V max, for the incision step. The approximate values of K m and V max for normal and XP variant are similar while for the heterozygotes of an XPA individual K m values are normal (around1 Jm −2), but there is only half the amount of normal enzyme activity. XPD and H cells express low levels of active enzyme, between 5 and 15% of that of the normal, but while the K m of KPH is very similar to that of normal cells, that of two XPD strains examined is between 2- and 3-fold higher.