Cell-cycle kinetics and ultraviolet light survival in UV-1, a Chinese hamster ovary cell mutant defective in post-replication recovery

0 Eleanor Roosevelt Institute for Cancer Research Inc. and Department of Radiology, University of Colorado Health Sciences Center , Denver, Colorado 80362 , U.S.A UV-i, an ultraviolet-sensitive mutant of CHO-Ki, is abnormally slow to recover from the inhibition of DNA synthesis caused by u.v. irradiation. When synchronized UV-I cells are irradiated in G1) their movement into S phase is unaltered, but thymidine incorporation is depressed (compared with that in the parent cell similarly treated). When irradiated in S phase, again incorporation is more depressed, and S phase suffers a greater delay in UV-i than in the parent cell. UV-i and its parent have similar capacities for excision repair of u.v.-induced damage inflicted in GlF and so enter S phase with similar amounts of unrepaired damage. The single-cell survival was measured after irradiation at different points in the cell cycle. The mutant and parent cells have similar values of Da (mean lethal dose) except in mitosis, when the parent cell shows markedly greater resistance to u.v. irradiation. ), (quasi-threshold dose) is fairly constant for the parent cell, but in UV-i it falls to a minimum in S phase. The responses of UV-i to u.v. irradiation are generally consistent with its known defect in the process of post-replication recovery, i.e. the ability to join up the abnormally small DNA fragments synthesized on a u.v.-damaged template. - SURVIVAL IN POST-REPLICATION RECOVERY ANDREW COLLINS1* AND CHARLES WALDREN1 UV-i is a mutant of the Chinese hamster cell, CHO-Ki, which was isolated by nylon-cloth replica-plating on the basis of its enhanced sensitivity to killing by ultraviolet (u.v.) light (Stamato & Waldren, 1977). u.v.-sensitive mutants are commonly defective in some aspect of the repair of u.v.-induced DNA damage (Thompson et al. 1980; Schultz, Trosko & Chang, 1981), and we found (Stamato, Hinkle, Collins & Waldren, 1981) that, although UV-i has a normal capacity for excision repair, it is defective in 'post-replication recovery'. The DNA synthesized by UV-i after u.v. irradiation (replicative rather than repair synthesis) has the same initial size as theDNA made by the parent cell; however, the conversion of nascent DNA to high molecular weight DNA is fourfold slower in UV-i (Stamato et al. 1981). This deficiency in postreplication recovery is associated with a diminished rate of mutagenesis, suggesting that post-replication recovery in normal cells is an error-prone process, which may be responsible for the production of mutations (Stamato et al. 1981). Address for reprint requests. The purpose of this report is to test the prediction that a defect in u.v. response specific to S phase will have effects on cell survival after u.v. irradiation that are related to the position of cells in the cell cycle at the time of irradiation. Using cells collected by metaphase arrest and released synchronously into interphase, we have irradiated UV-i and its parent cell at intervals during the cycle, and measured the single-cell plating efficiency. We have also examined the effect of u.v. irradiation at different times in the cycle on the overall rate of DNA synthesis and on the transit of cells through S phase, and we have compared rates of enzymic incision at DNA damage sites in synchronized cells. Our findings substantiate the characterization of the lesion in UV-i, and throw some light on the relationship in normal cells between repair, survival, and the cell cycle. MATERIALS AND METHODS Cell culture and synchronization The cell lines studied were 772-56 (a glycine- and proline-requiring mutant derived from CHO-Ki) and UV-i, isolated from 772-56 as a u.v.-sensitive mutant (Stamato & Waldren, 1977). They were cultured in F12 medium (Ham, 1965), without hypoxanthine or thymidine, and with 5 % serum (3:1, newborn calf: foetal calf serum). They normally grew in plastic dishes, but for synchronization they were cultured as monolayers in rotating glass bottles. An 8-h incubation with 2'5 x io~s M-thymidine was followed by 10 h in fresh medium, with colcemid (0-05 /ig/ml) present during the final 4 h. Mitotic cells (purity at least 90 %) were collected in suspension by gentle agitation of the bottles, centrifuged, washed twice with warm (37 C) phosphate-buffered saline (PBS), and either used at once in experiments examining mitotic cells, or centrifuged, resuspended in growth medium, and plated out in dishes to progress to the required stage of the cell cycle. In all but survival experiments (see below), cells were irradiated in titu in dishes, after removing growth medium. The source was a germicidal lamp emitting mainly at 254 nm and the exposure rate was either 0-4 or i-o Jm~* s"1. Medium was then replaced. Control cells were mock-irradiated. Monitoring DNA synthesis The time-course of DNA synthesis in random or synchronous cultures was determined by pulsing samples of cells at intervals with [wef/i}>/-sH]thymidine (Radiochemical Centre, Amersham) as described in the figure legends. After 30 min incubation, labelled medium was removed, the cells washed in cold saline (0-9% NaCl) and either: (1) fixed with Carnoy's fixative (3 : i , v/v, methanol .-glacial acetic acid), extracted several times with cold (4 C) 5 % (w/v) trichloroacetic acid, and prepared for autoradiography as described (Collins & Johnson, J979); or (2) lysed in 1 ml 0-5 M-NaOH, acidified with 1 ml 20% (w/v) trichloroacetic acid, and macromolecules collected on Whatman GF/C glass-fibre filters for scintillation counting of 'H incorporated into DNA. Measuring excision repair-related DNA break accumulation Cells in rotating bottles were incubated for 24 h with [!H]thymidine (55 Ci/mmol, O'l /iCi/ml) to prelabel DNA, and then synchronized as above. Mitotic cells were plated out at 10s per 35-mm dish. They were irradiated with u.v. (10 Jm"1) during the early part of the cell cycle, and incubated with DNA synthesis inhibitors hydroxyurea (HU; io~* M) and i-/?-D-arabinofuranosylcytosine (araC; io~* M) (both from Sigma), as described in Results. After incubation, cells were washed with cold saline, and 1 ml alkaline sucrose (5 % sucrose, o-oi M-EDTA, o-3M-NaCl, 0-15 M-NaOH) was added to each dish. Alkaline lysis continued for 20 min at 4 C, and was ended by addition of 0-2 ml 1 M - K H , P O 4 . DNA was sheared by passing through a 25-gauge needle, the sample diluted to 20 ml with 0-04% sodium dodecyl sulphate in water, and the DNA separated by hydroxyapatite chromatography into native and denatured fractions (Collins, 1977). The extent of alkaline denaturation of prelabelled DNA depends on the frequency of DNA breaks introduced by repair enzymes at sites of u.v. damage. Results are shown in terms of DNA breaks per 10' daltons, according to a calibration of the lysis solution using cells X-irradiated to produce known break frequencies (Squires, Johnson & Collins, 1982). Measuring u.v. survival Mitotic or interphase cells (trypsinized at 3-ho (...truncated)