Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2008 Sep 1.
Published in final edited form as: DNA Repair (Amst). 2007 Jun 5;6(9):1380–1385. doi: 10.1016/j.dnarep.2007.04.005

X-rays induce distinct patterns of somatic mutation in fetal versus adult hematopoietic cells

Li Liang *, Li Deng *, Marc S Mendonca †, Yanping Chen *, Betty Zheng *, Peter J Stambrook ‡, Changshun Shao *, Jay A Tischfield *,
PMCID: PMC2063444  NIHMSID: NIHMS29734  PMID: 17553756

Abstract

There are a variety of mechanisms and pathways whereby cells safeguard their genomes in the face of environmental insults that damage DNA. Whether each of these pathways is equally robust at specific developmental stages in mammals and whether they are also modulated in a tissue-specific manner, however, are unclear. Here we report that ionizing radiation (IR) produces different types of somatic mutations in fetal cells compared with adult cells of the same lineage. While 1 Gy of X-ray significantly induced intragenic point mutations in T cells of adult mice, no point mutational effect was observed when applied to fetuses. Fetal exposure to IR, on the other hand, led to a significant elevation of mitotic recombination in T cells, which was not observed in adults. Base excision repair (BER) activity was significantly lower in fetal hematopoietic cells than in adult cells, due to a low level of DNA polymerase β, the rate-limiting enzyme in BER. In fetal hematopoietic cells, this low BER activity, together with a high rate of proliferation, causes X-ray-induced DNA lesions, such as base damage, single strand breaks and double strand breaks to be repaired by homologous recombination, which we observe as mitotic recombination. Higher BER activity and a relatively lower rate of cell proliferation likely contribute to the significant induction of DNA point mutations in adults. Thus, the mutational response to IR is at least partly determined by the availability of specific repair pathways and other developmentally-regulated phenotypes such as mitotic index.

Prenatal irradiation with low doses of X-rays imposes a serious risk toward developing childhood leukemia [1]. Both human and animal studies have demonstrated that exposure to IR during the late fetal period causes the greatest induction of cancers [1]. Furthermore, the types of cancer induced by IR exposure in utero are different from those seen in adults. Sasaki and Kasuga [2] reported that B6C3F1 mice exposed to X-rays on day 17 post-conception developed solid tumors, but not leukemia. In contrast, irradiation of young adults (15 weeks of age) was associated with development of myeloid leukemia. These differences in the type of cancer induction by IR at different developmental stages suggest the presence of tissue- and/or developmental stage-specific response factors. The mechanisms underlying developmentally modulated IR-induced carcinogenesis are poorly understood. Since there is a clear linkage between DNA repair, mutagenesis and cancer, we propose that cellular responses to IR-induced DNA damage may differ between developmental stages. In the present study, we asked whether mutational events that predominate at the autosomal Aprt (adenine phosphoribosyltransferase) and X-linked Hprt (hypoxanthine-guanine phosphoribosyltransferase) loci in T-cells of mice exposed to IR as adults are the same or different from those in T-cells of mice that had been exposed in utero. We showed that IR exposure at different developmental stages generated distinct mutational spectra in T-cells.

Materials and methods

Animals and irradiation

Pregnant C57BL/6 mice that had been mated to C3H males (thus, carrying B6C3F1 fetuses) were subjected to whole body irradiation on days 16−18 of gestation with a single dose of 0.5, 1, or 2 Gy of X-rays. Offspring of irradiated animals were raised under controlled conditions of light and temperature. Two-month old adult mice were also subjected to a single 1 Gy dose of whole body X-irradiation. The Aprt genotype of the B6C3F1 mice was determined by allele-specific PCR [3].

Isolation of 2,6-diaminopurine-resistant (DAPr) and 6-thioguanine-resistant (6-TGr) T lymphocytes and characterization of mutant clones.

At specific times following X-irradiation (20, 40, or 60 days), groups of the B6C3F1 Aprt+/- mice were sacrificed and DAPr and 6-TGr splenic T cell colonies were selected, as previously described [3-5].

Molecular and cytogenetic characterizations of T cell clones are described elsewhere [3;4].

Nuclear protein extracts isolated from fetal and adult tissues

Fetal spleens were collected on day 16 of gestation and macerated in ice-cold RPMI-1640 medium. Spleens and thymuses from adult mice (two months old) were also crushed in ice-cold RPMI-1640 medium. Spleen or thymus cell suspensions were passed through cell strainers (70 μm) and harvested by centrifugation. Adult bone marrow cells were obtained by flushing the medullary cavities of femurs with ice-cold RPMI-1640 medium using a 25-gauge needle. Nuclear protein extracts were prepared as described [6].

In vitro analysis of base excision repair (U:G mismatch repair assay)

An in vitro base excision repair (BER) assay that measures the short patch BER efficiency of cell extracts was used [7]. Briefly, a 30-mer oligonucleotide (5'-GAGCCGGCACTGGUGCCCAGCTGATATCGC-3') containing a uracil at position 14 was annealed to the oligonucleotide (5'-GCGATATCAGCTGGGCGCCAGTGCCGGCTC-3'). The corresponding “normal” duplex containing a CG base pair was used as control. The DNA duplex was incubated in a reaction mixture (25 μl) with an equivalent amount (6 μg) of nuclear extract from mouse fetal or adult cells at 37°C for 30 min. The reaction mixture contained 40 mM phosphocreatine di(tris) salt, 5 mM MgCl2, 1 mM dithiothreitol, 2 mM ATP, 20 μM each of dATP, dGTP, dTTP and dCTP, 1 μCi [α-32P]dCTP, 2.5 μg creatine phosphokinase, 50 mM NaCl, and 0.5 μg 30 bp duplex oligoncleotide (UG or CG substrate). Reactions were stopped by proteinase K treatment (1 μl of 10 mg/ml proteinase K per reaction) at 37°C for 10 min. Samples were loaded onto a 15% polyacrylamide gel and electrophoresed at 150 V for 60 min. The BER activity is determined by the incorporation of the [α-32P]dCTP, in replacing the uracil in the 30 bp DNA duplex. The radiolabeled 30 bp fragment was visualized and quantified with a Typhoon PhosphoImager using ImageQuant software (Molecular Dynamics).

Immunoblotting

Equivalent amounts of nuclear extracts from fetal and adult cells were separated with SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. Membranes were blocked at room temperature for 30 min in blocking buffer (3% non-fat dry milk in PBS containing 0.1% Tween 20 (T-PBS)) and incubated at 4°C overnight with primary antibody diluted in blocking buffer. After five washes with T-PBS, membranes were incubated at room temperature for 1 hr with secondary antibody diluted in blocking buffer. Immunoblots were visualized by enhanced chemiluminescence. Primary antibodies used in this study were: mouse anti-DNA polymerase β (Lab Vision corporation, Fremont, CA), and rabbit anti-β-actin (Abcam, Cambridge, MA).

Results

Prenatal X-irradiation increases the Aprt but not the Hprt mutant frequency in T cells

We and others have observed that a single dose of 1 Gy X-irradiation given to mice at two months of age significantly increases the mutant frequency (MF) at Hprt in T cells, but does not significantly increase the MF at the Aprt locus in the same cell type [8;9]. Specifically, 1 Gy X-ray increased the Hprt MF by 3.3-fold as compared to unirradiated controls (11 × 10−6 v.s. 3.3 × 10−6, in irradiated mice and controls, respectively) [8]. PCR amplification of Hprt indicated that intragenic deletions in Hprt were significantly induced by X-irradiation (Nguyen, Liang and Tischfield, unpublished data). Molecular analyses of Aprt mutants also demonstrated that intragenic alterations in Aprt (base substitutions and deletions) but not large chromosome alterations were induced by 1 Gy X-ray [8]. However, the putative induction in intragenic alterations (3.2 × 10−6) at Aprt was not enough to produce a significant increase in overall Aprt MF due to the high spontaneous Aprt MF (24 × 10−6). Thus, X-irradiation induces intragenic alterations at both loci.

We hypothesized that fetal cells respond to X-rays differently than adult cells and, as a consequence, harbor a different spectrum of somatic mutations after exposure. To test this hypothesis, we exposed fetuses to X-rays and characterized the mutations using Hprt and Aprt as reporters. Pregnant C57BL/6 mice carrying B6C3F1 fetuses were irradiated with a single dose of 1 Gy X-rays on days 16 − 18 of gestation and the B6C3F1 Aprt+/- mice were sacrificed at 20, 40 or 60 days following irradiation. As shown in Figure 1A, the spontaneous Aprt and Hprt MFs in T cells of control mice were unchanged with maturation. The Aprt MF in T cells from mice irradiated in utero was about two-fold greater than that of controls at day 40 post exposure, and remained at a high level at day 60. This time-dependent increase in the Aprt MF after exposure may be attributed to a combination of the time required for the phenotypic expression of 2,6-diaminopurine resistance and the time required for the migration of mutants from hematopoietic tissues to the spleen and peripheral lymphatic tissues. In contrast to the post-exposure time-dependent increase in the Aprt MF, the Hprt MF in T cells of mice irradiated in utero was unchanged compared with control values, at all time points tested (Fig. 1A).

Figure 1. Prenatal X-irradiation increases the Aprt but not the Hprt mutant frequency in T cells.

Figure 1

Open in a new tab

A. Time course of mutagenic effects of X-rays on T-cells of B6C3F1 mice. Pregnant C57BL/6 mice carrying B6C3F1 fetuses were irradiated with a single dose of 1 Gy X-rays on days 16 − 18 of gestation. T cells were isolated from irradiated offspring and assayed for mutations at Hprt and Aprt at 20, 40 and 60 days after exposure. Each point represents the mean mutant frequency (N = 5 − 22 mice/group). The error bar represents the standard error of the mean. B. Effect of X-ray dose on the Aprt and Hprt MFs in T-cells from B6C3F1 mice irradiated in utero. B6C3F1 fetuses were irradiated with a single dose of 0.5, 1, or 2 Gy of X-rays on days 16 − 18 of gestation. T cells were isolated 60 days after prenatal exposure. Each point represents the mean mutant frequency (N = 5 − 22/group). The error bar shows standard error of the mean.

To determine whether the T cell MF displays a dose-dependent response at the Aprt and Hprt loci of mice treated in utero, pregnant mice carrying B6C3F1 fetuses were irradiated with a single 0.5 Gy, 1.0 Gy, or 2.0 Gy dose of X-rays on days 16 − 18 of gestation. The mice were sacrificed 60 days later and T-cells were isolated and placed into culture in appropriate selection media. As shown in Figure 1B, the mean Aprt MF of all treated groups is significantly higher than that of the control (P=0.004, one way ANOVA). However, the dose-response curve does not show linearity with increasing dose. The curve peaks at 1 Gy (44 × 10−6) and drops at 2 Gy (32 × 10−6). Interestingly, in T-cells from irradiated mice, the MF at Hprt remained flat with dose and was not elevated over that of the unirradiated mice (P=0.62, one way ANOVA, Fig. 1B).

Thus, irradiation of adult T-cells with 1 Gy of X-rays significantly elevated the MF at the Hprt locus, but not at Aprt. In fetal cells, however, the same dose of X-rays had the opposite effect, increasing mutations at the Aprt locus, but not at Hprt.

Mitotic recombination (MR) is the major mechanism for the X-ray-induced Aprt mutants in the fetus

To determine the types of mutational events that contributed to the increased MF at Aprt in T cells of mice exposed to 1 Gy X-rays in utero, we characterized the Aprt mutant T cells (DAPr T cells). Based on molecular and cytogenetic analyses, the frequency with which variants arose as a consequence of MR was calculated to be 2.6 fold higher than that of unirradiated controls (37 × 10−6 vs. 14 × 10−6) (Fig. 2). The frequency of variants derived from interstitial deletion/gene conversion was also increased although these mutational events were still rare (Fig. 2). DNA sequencing, however, indicated that the frequency of variants caused by intragenic point mutations, including small scale deletions, insertions, and base substitutions, was not increased (Fig. 2). These data are consistent with our observation that the frequency of Hprt mutants, which are primarily caused by intragenic point mutations, is not increased when mice are exposed in utero. In contrast, the frequency of intragenic point mutations was significantly increased when mice were irradiated with 1 Gy X-rays at two months of age (Fig. 2). Thus, these results clearly demonstrate that X-irradiation produces different types of mutations in fetal cells than in adult cells of the same lineage.

Figure 2. Mitotic recombination, but not point mutation, is the major cause of the increased Aprt MF in T cells from mice irradiated in utero.

Figure 2

Open in a new tab

A minimum of 120 DAPr colonies per treatment group were characterized as described in the Methods and Materials. The value of each bar represents the Aprt MF caused by a specific mutation type and is calculated by multiplying the Aprt MF by the fraction of each given mutation type.

Base excision repair activity is lower in mouse fetal hematopoietic cells than in adult hematopoietic cells.

The base excision repair (BER) pathway repairs altered bases and DNA single-strand breaks induced by IR [10]. We hypothesized that the absence of X-ray induced point mutations at the fetal stage was caused by higher BER activity. To test this proposition, we measured BER efficiency at different developmental stages in nuclear extracts prepared from tissues that harbor hematopoietic stem and precursor cells. We isolated nuclear extracts from bone marrow, spleen and thymus cells of adult mice as well as fetal spleen cells, since the major site of hematopoiesis switches from the fetal liver to the spleen in late fetal development [11]. Surprisingly, fetal spleen cells displayed the lowest BER activity, about 5-fold lower than cells from adult bone marrow, spleen or thymus (Fig. 3A). Since DNA polymerase β (Pol β) has been reported to be the rate-limiting enzyme in the BER pathway [12], responsible for both repair synthesis and excision of the deoxyribose phosphate moiety generated during the repair process, we measured the Polβ protein level by Western blot. As shown in Figure 3B, the protein level of Pol β in fetal spleen cells is much lower than that in adult cells. Thus, the lack of X-ray-induced point mutations at the fetal stage is not a consequence of elevated BER.

Figure 3. Fetal hematopoietic cells have a lower level of DNA polymerase β-dependent BER activity than adult hematopoietic cells.

Figure 3

Open in a new tab

A. In vitro BER activity in extracts of cells from fetal and adult mice. BER reactions contained the 30 bp DNA duplex with a UG mismatch, [α-32P]dCTP, and nuclear extracts. Replacement by BER of an unpaired U with a paired C [α-32P]dCTP produces a radioactive 30 bp band. The corresponding “normal” duplex containing the CG base pair served as a control. The radiolabeled 30 bp fragment was visualized and quantified with a Typhoon PhosphoImager using ImageQuant software. B. Western blot analysis of DNA polymerase β in extracts of fetal and adult cells.

It has been reported that cells with BER deficiency exhibit increased homologous recombination (HR) activity that is reflected by an increased frequency of sister chromatid exchange and increased Rad51 focus formation [13;14]. Our finding that X-rays induced MR, but not point mutation, in fetal hematopoietic tissues is consistent with these reports that BER deficiency can be accompanied by an increase in HR.

Discussion

We have shown that adult mice exposed to 1 Gy of X-rays display a significant elevation of intragenic alterations (including base substitutions and deletions/insertions) at Aprt in T cells (Fig. 2). In contrast, when the same dose was administered to fetuses, MR, but not intragenic alteration, was significantly induced (Fig. 2). Thus, the same DNA damaging agent induced different kinds of somatic mutations following X-ray exposure of adults or fetuses. This developmental stage-specific induction of either MR or intragenic point mutations may reflect preferential use of different DNA repair pathways at distinct developmental stages.

Ionizing radiation causes various types of DNA damage, including single-strand DNA breaks (SSBs), double-strand DNA breaks (DSBs), and base damage (BD) [15]. BD and SSB are primarily repaired by the BER pathway [10]. However, BER can lead to base substitutions since Pol β, a key enzyme in BER, lacks proofreading activity [16]. DSBs are repaired by two distinct types of repair pathways, homologous recombination repair (HR) and non-homologous end-joining (NHEJ). NHEJ can be mutagenic, resulting in deletions and insertions at the DSB site. Although repair by HR is presumed to be precise, it has the potential to result in loss of heterozygosity when recombination occurs between homologous chromosomes with a heterozygous locus distal to the crossover.

In the absence of BER, HR plays an important role in the repair of BD and SSB in mammalian cells, since stalled and collapsed DNA replication forks at unrepaired apurinic sites and SSB sites serve as a signal for recombination repair [17;18]. It was reported recently that lack of Pol β-dependent BER activity leads to SSB accumulation in G1 phase [13]. Cells entering into S phase with unrepaired SSBs exhibit a rapid phosphorylation of histone H2AX, indicating that the unrepaired SSBs have been converted to DSBs. This is followed by the relocalization of Rad51 protein that is essential for HR [13]. The lower level of BER activity in mouse fetal hematopoietic cells compared to their adult counterparts (Fig. 3A), due to diminished Pol β protein (Fig. 3B), suggests that BDs and SSBs may not be repaired efficiently by BER in fetal hematopoietic cells. Such lesions could produce stalled or collapsed replication forks, and then be repaired by HR if they persist into S phase.

The preferential induction of MR as a consequence of prenatal exposure to X-rays could also be attributable to a higher frequency of target cells in S/G2 phase. During fetal development, hematopoietic cells, including hematopoietic stem cells (HSCs), multiply dramatically to keep pace with growth. Indeed, fetal HSCs proliferate more rapidly than those in adult bone marrow [19]. On the other hand, most mature T lymphocytes are in G0 phase in vivo until they are activated by specific mitogens or antigens [20]. Thus, it is reasonable to assume that the fraction of fetal hematopoietic cells in S-phase is greater than that of adult cells. Since the choice of repair pathways for DSBs, HR and/or NHEJ, is dependent on the phase of cell cycle when DSBs occur, with HR predominant during the S and G2 phases and NHEJ in G0/G1 phase [21], more actively proliferating cells, such as those in the fetus, are more likely to use HR to repair DSBs.

Therefore, a low level of BER coupled with high proliferative activity in fetal hematopoietic cells results in HR as the predominant pathway for the repair of X-ray-induced DNA damage, including BDs, SSBs, and DSBs. In contrast, BER and NHEJ may be the major repair pathways for X-ray-induced lesions in less actively dividing adult cells. The fact that HR, NHEJ and BER produce different mutagenic effects explains the characteristic induction of MR or intragenic point mutations at specific developmental stages. Our finding that cells of the same lineage at different developmental stages use distinctive DNA repair mechanisms, some of which are error prone, may partially explain why different developmental stages are more susceptible to specific cancers. The genetic risk(s) of prenatal exposure to IR should be reevaluated in light of this new concept.

Acknowledgements

This work was supported by grants from the National Institutes of Health (ES011633 and P30ES05022), the National Aeronautics and Space Administration (NNG05GN24G), and a New Jersey Stem Cell Research grant from New Jersey Commission on Science and Technology.

Glossary

Abbreviations:

Aprt

adenine phosphoribosyltransferase

BER

base excision repair

DSB

double strand break

Hprt

hypoxanthine-guanine phosphoribosyltransferase

IR

ionizing radiation

MF

mutant frequency

MR

mitotic recombination

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reference List

  • 1.ICRP Biological Effects after Prenatal Irradiation (Embryo and Fetus). ICRP Publication. Ann ICRP. 2003;90 [PubMed] [Google Scholar]
  • 2.Sasaki S, Kasuga T. Life shortening and carcinogenesis in mice irradiated at perinatal period with gamma rays. In: Thompson RC, Mahaffey JA, editors. Lifespan Radiation Effects Studies in Animals: What Can They Tell Us? National Technical Information Service; Springfield, USA: 1986. pp. 357–367. [Google Scholar]
  • 3.Shao C, Deng L, Henegariu O, Liang L, Raikwar N, Sahota A, Stambrook PJ, Tischfield JA. Mitotic recombination produces the majority of recessive fibroblast variants in heterozygous mice. Proc.Natl.Acad.Sci.U.S.A. 1999;96:9230–9235. doi: 10.1073/pnas.96.16.9230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Liang L, Deng L, Shao C, Stambrook PJ, Tischfield JA. vivo loss of heterozygosity in T-cells of B6C3F1 Aprt(+/-) mice. Environ.Mol.Mutagen. 2000;35:150–157. [PubMed] [Google Scholar]
  • 5.Shao C, Yin M, Deng L, Stambrook PJ, Doetschman T, Tischfield JA. Loss of heterozygosity and point mutation at Aprt locus in T cells and fibroblasts of Pms2-/-mice. Oncogene. 2002;21:2840–2845. doi: 10.1038/sj.onc.1205358. [DOI] [PubMed] [Google Scholar]
  • 6.Liang L, Deng L, Chen Y, Li GC, Shao C, Tischfield JA. Modulation of DNA end joining by nuclear proteins. J.Biol.Chem. 2005;280:31442–31449. doi: 10.1074/jbc.M503776200. [DOI] [PubMed] [Google Scholar]
  • 7.Nealon K, Nicholl ID, Kenny MK. Characterization of the DNA polymerase requirement of human base excision repair. Nucleic Acids Res. 1996;24:3763–3770. doi: 10.1093/nar/24.19.3763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Liang L, Mendonca MS, Deng L, Nguyen SC, Shao C, Tischfield JA. Reduced Apoptosis and Increased Deletion Mutations at Aprt Locus In vivo in Mice Exposed to Repeated Ionizing Radiation. Cancer Res. 2007;67:1910–1917. doi: 10.1158/0008-5472.CAN-06-1476. [DOI] [PubMed] [Google Scholar]
  • 9.Wijnhoven SW, Van Sloun PP, Kool HJ, Weeda G, Slater R, Lohman PH, van Zeeland AA, Vrieling H. Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse. Proc.Natl.Acad.Sci.U.S.A. 1998;95:13759–13764. doi: 10.1073/pnas.95.23.13759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Krokan HE, Nilsen H, Skorpen F, Otterlei M, Slupphaug G. Base excision repair of DNA in mammalian cells. FEBS Lett. 2000;476:73–77. doi: 10.1016/s0014-5793(00)01674-4. [DOI] [PubMed] [Google Scholar]
  • 11.Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc.Natl.Acad.Sci.U.S.A. 1995;92:10302–10306. doi: 10.1073/pnas.92.22.10302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Srivastava DK, Berg BJ, Prasad R, Molina JT, Beard WA, Tomkinson AE, Wilson SH. Mammalian abasic site base excision repair. Identification of the reaction sequence and rate-determining steps. J.Biol.Chem. 1998;273:21203–21209. doi: 10.1074/jbc.273.33.21203. [DOI] [PubMed] [Google Scholar]
  • 13.Pascucci B, Russo MT, Crescenzi M, Bignami M, Dogliotti E. The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells. Nucleic Acids Res. 2005;33:280–288. doi: 10.1093/nar/gki168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sobol RW, Kartalou M, Almeida KH, Joyce DF, Engelward BP, Horton JK, Prasad R, Samson LD, Wilson SH. Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses. J.Biol.Chem. 2003;278:39951–39959. doi: 10.1074/jbc.M306592200. [DOI] [PubMed] [Google Scholar]
  • 15.Ward JF. Complexity of damage produced by ionizing radiation. Cold Spring Harb.Symp.Quant.Biol. 2000;65:377–382. doi: 10.1101/sqb.2000.65.377. [DOI] [PubMed] [Google Scholar]
  • 16.Osheroff WP, Jung HK, Beard WA, Wilson SH, Kunkel TA. The fidelity of DNA polymerase beta during distributive and processive DNA synthesis. J.Biol.Chem. 1999;274:3642–3650. doi: 10.1074/jbc.274.6.3642. [DOI] [PubMed] [Google Scholar]
  • 17.Helleday T. Pathways for mitotic homologous recombination in mammalian cells. Mutat.Res. 2003;532:103–115. doi: 10.1016/j.mrfmmm.2003.08.013. [DOI] [PubMed] [Google Scholar]
  • 18.McGlynn P, Lloyd RG. Recombinational repair and restart of damaged replication forks. Nat.Rev.Mol.Cell Biol. 2002;3:859–870. doi: 10.1038/nrm951. [DOI] [PubMed] [Google Scholar]
  • 19.Rebel VI, Miller CL, Eaves CJ, Lansdorp PM. The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their adult bone marrow counterparts. Blood. 1996;87:3500–3507. [PubMed] [Google Scholar]
  • 20.O'Neill JP, Nicklas JA, Hunter TC, Batson OB, Allegretta M, Falta MT, Branda RF, Albertini RJ. The effect of T-lymphocyte 'clonality' on the calculated hprt mutation frequency occurring in vivo in humans. Mutat.Res. 1994;313:215–225. doi: 10.1016/0165-1161(94)90052-3. [DOI] [PubMed] [Google Scholar]
  • 21.Valerie K, Povirk LF. Regulation and mechanisms of mammalian double-strand break repair. Oncogene. 2003;22:5792–5812. doi: 10.1038/sj.onc.1206679. [DOI] [PubMed] [Google Scholar]

RESOURCES