The Fate of DNA–protein Crosslinks Formed in γ-Irradiated Metaphase Cells

Abstract

The induction of DNA-protein crosslinks (DPC) was compared in gamma-irradiated metaphase and asynchronous Chinese hamster V79 cells. Unirradiated metaphase cells were found to have a higher level of background DPC than unirradiated asynchronous cells, and the metaphase cells were less susceptible to radiation-induced DPC production than were asynchronous cells. SDS-PAGE analysis of crosslinked proteins prepared from the two cell populations, both irradiated and unirradiated, showed very similar protein patterns. Crosslinked DNA was isolated and probed with radioactively labelled interphase poly(A+)RNA. The results indicated that the hypersensitivity of interphase actively transcribing DNA sequences to radiation-induced DPC formation was maintained at metaphase when the chromosomes are highly condensed. In contrast to asynchronous cells, radiation-induced DPC formed in metaphase cells were not removed during a 4 h post-irradiation period. However, metaphase cells appear to be able to remove the active DNA involved in DPC as indicated by a depletion of the probed sequences in the unrepaired DPC. Cell size analysis as well as cytological examination of the irradiated metaphase cells showed an absence of cell division during post-irradiation incubation. Furthermore, about 50% of the irradiated metaphase cells grew into giant cells which contain multiple nuclei and micronuclei, an indication of aberrant chromosome segregation.