UV Radiation Induces Delayed Hyperrecombination Associated with Hypermutation in Human Cells

Abstract

Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an ∼5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m²), revealed as mixed GFP^+/− colonies. UV-B did not induce DHR at an equitoxic (75 J/m²) dose or a higher dose (150 J/m²). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were ∼5-fold higher in strains derived from GFP^+/− (DHR) colonies than in strains in which recombination was directly induced by UV (GFP⁺ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP⁺ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP^+/− strains were small (1- to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Melinda Wilson for supplying cell lines, Rebecca Lee and Genevieve Phillips for assistance with fluorescence microscopy, and Howard Liber for helpful comments. Images were generated in the UNM Cancer Center Fluorescence Microscopy Facility, which received support from NCRR S10 RR14668, NSF MCB9982161, NCRR P20 RR11830, NCI R24 CA88339, NCRR S10 RR19287, NCRR S10 RR016918, and NCI P30 CA118100 awards. We acknowledge the UNM Shared Flow Cytometry Resource, which is supported in part by the NCI P30 CA118100 award.

This work was supported by the Biological and Environmental Research Program (BER), U.S. Department of Energy, grant DE-FG02-01-ER63230, and National Cancer Institute (NIH) grant R01CA73924 to W.F.M., NCI grant R21CA113687 to G.S.T., and NCI grant R01CA77693 to J.A.N.