DNA Repair 4 (2005) 782–792
Inﬂuence of double-strand-break repair pathways on radiosensitivity
throughout the cell cycle in CHO cells
John M. Hinz, N. Alice Yamada, Edmund P. Salazar,
Robert S. Tebbs
, Larry H. Thompson
Biosciences Directorate, L441 Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808, USA
Received 3 January 2005; received in revised form 17 March 2005; accepted 22 March 2005
Unrepaired DNA double-strand breaks (DSBs) produced by ionizing radiation (IR) are a major determinant of cell killing. To determine
the contribution of DNA repair pathways to the well-established cell cycle variation in IR sensitivity, we compared the radiosensitivity of
wild-type CHO cells to mutant lines defective in nonhomologous end joining (NHEJ), homologous recombination repair (HRR), and the
Fanconi anemia pathway. Cells were irradiated with IR doses that killed ∼90% of each asynchronous population, separated into synchronous
fractions by centrifugal elutriation, and assayed for survival (colony formation). Wild-type cells had lowest resistance in early G1 and highest
resistance in S phase, followed by declining resistance as cells move into G2/M. In contrast, HR-defective cells (xrcc3 mutation) were most
resistant in early G1 and became progressively less resistant in S and G2/M, indicating that the S-phase resistance in wild-type cells requires
HRR. Cells defective in NHEJ (dna-pk
mutation) were exquisitely sensitive in early G1, most resistant in S phase, and then somewhat less
resistant in G2/M. Fancg mutant cells had almost normal IR sensitivity and normal cell cycle dependence, suggesting that Fancg contributes
modestly to survival and in a manner that is independent of cell cycle position.
Published by Elsevier B.V.
Keywords: Homologous recombination; Nonhomologous end joining; Fanconi anemia; DNA double-strand breaks; Cell synchrony
Ionizing radiation (IR) produces a multitude of DNA oxi-
dized and ring-opened bases, as well as single- and double-
strand breaks (DSB), by direct ionization of DNA or as
indirect base and backbone damage caused by highly reac-
tive hydroxyl radicals generated by the ionization of water
molecules [1,2]. Additional breaks of both types result from
the processing of these lesions by DNA repair and during
DNA replication [3,4]. The degree of cell killing after IR
exposure generally correlates with the number of unrepaired
Abbreviations: CHO, Chinese hamsterovary; HRR,homologousrecom-
bination repair; NHEJ, nonhomologous end joining; IR, ionizing radiation;
FA, Fanconi anemia; DSB, double-strand break
Corresponding author. Tel.: +1 925 4225658; fax: +1 925 4222099.
E-mail address: email@example.com (L.H. Thompson).
Present address: Applied Biosystems, 850 Lincoln Centre Drive, Bldg
200, Foster City, CA 94404, USA.
DSBs [5,6]. Cells have two genetically deﬁned pathways for
repairing DSBs. The error-prone nonhomologous end join-
ing (NHEJ) pathway rapidly and promiscuously ligates the
ends of broken chromosomes while homologous recombina-
tion repair (HRR) uses an homologous template in a sister
chromatid or homologous chromosome to perform error-free
Major constituents of the NHEJ pathway are the DNA-
dependant protein kinase holo-enzyme (composed of the
DNA end–binding Ku70/Ku80 dimer and the catalytic sub-
unit DNA-PKcs) and the XRCC4–LIG4 complex respon-
sible for ligation [10–12]. HRR is performed by proteins
that are conserved among vertebrates, and to a lesser extent
between mammals and yeast: RPA, Rad51, Rad52, Rad54,
and the Rad51 paralogs (XRCC2/3, Rad51B/C/D) [13–15].
The importance of both DSB-repair pathways in cell survival
after IR exposure is revealed by the pronounced radiosen-
sitivity of mutant cells (with the exception of Rad52), but
1568-7864/$ – see front matter. Published by Elsevier B.V.