A mechanism for relief of replication blocks by activation of unused origins and age-dependent change in the caffeine susceptibility in xeroderma pigmentosum variant

Abstract

The first XP16KO-I (age 42 without cancer) and the second XP16KO-II (age 52 after carcinogenesis) skin fibroblasts of a xeroderma pigmentosum variant (XPV) were studied. First, caffeine had no effect on XP16KO-I cells, but it potentiated UV killing and inhibited the recovery of DNA synthesis and the elongation of nascent DNA after UV irradiation in XP16KO-II cells, indicating an age-dependent change from caffeine resistance to sensitivity. These results confirmed a previous similar change in an unrelated XPV subject and the existence of 2 XPV subgroups, caffeine-sensitive and -resistant. Further, processing of post-excision breaks was also slightly more defective in XP16KO-II than in XP16KO-I cells. Second, nascent DNA pulse-labeled at 1.5 h after 5 J/m2 indicated an initial complete blockage of elongation by each dimer in XP16KO-II cells and less complete blocks in XP16KO-I and normal cells. The nascent DNA in 5 J/m2-irradiated XP16KO-II cells was 4 microns (8 x 10(6) Da) long for the peak fraction, with a possible range up to 20 microns of average replicon size, indicating activation of at least 4 microns-spaced new origins around 1.5 h post irradiation. Post-labeling chase without caffeine allowed nearly normal elongation to the present maximum molecular weight of 2.60-2.80 x 10(8) in 3-4 h in irradiated XP16KO-I and -II cells. Thus, the activation of unused origins at shorter spaces is a main mechanism for relief and the reduction of almost all unreplicated regions or blocks initially formed by 2 dimers in trans on both leading strands between converging forks. Post-labeling addition of 1 mM caffeine increased perpetuated blocks to a frequency of about 10% of the initial number of dimers in 4 h in XP16KO-II cells, but not in XP16KO-I and normal cells.