Nucleic Acids Research, 2001, Vol. 29, No. 19 3975-3981
© 2001 Oxford University Press
Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA
Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, USA
Received June 25, 2001; Revised and Accepted August 13, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we stably transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. Cells were then electroporated with a plasmid expressing endonuclease I-SceI to induce a DSB, and clones that had lost tk function were selected. In a previous study of DSB-induced tk-deficient clones, we found that
8% of recovered tk mutations involved the capture of one or more DNA fragments at the DSB site. Almost half of the DNA capture events involved the I-SceI expression plasmid, and several events involved retrotransposable elements. To learn whether only certain DNA sequences or motifs are efficiently captured, in the current work we electroporated an I-SceI expression plasmid along with HaeIII fragments of 
X174 genomic DNA. We report that 18 out of 132 tk-deficient clones recovered had captured DNA fragments, and 14 DNA capture events involved one or more fragments of X174 DNA. Microhomology existed at most junctions between X174 DNA and genomic sequences. Our work suggests that virtually any extrachromosomal DNA molecule may be recruited for the patching of DSBs in a mammalian genome. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In eukaryotic cells, DNA double-strand breaks (DSBs) may be repaired by homologous recombination or non-homologous end-joining (NHEJ) (1–12). During DSB repair via homologous recombination, the broken DNA sequence interacts with a homologous donor sequence and genetic information is exchanged between identical or nearly identical DNA sequences. Homologous recombination is considered to be an accurate mode of repair. NHEJ is accomplished by the joining of DNA ends with no interaction between the broken molecule and a donor sequence. In NHEJ there is no requirement for homology at the DNA termini being joined, although NHEJ may be facilitated by short terminal homologies (2). NHEJ may be accompanied by the deletion or gain of genetic material prior to healing of the DSB.
DSB repair mechanisms generally tend to produce little genetic change. For example, DSB-induced homologous recombination is often accomplished by gene conversion without associated crossovers (7,9,13,14) and NHEJ is typically accompanied by loss (or gain) of only a few nucleotides. Indeed, precise ligation of DSBs with no alteration of sequences may be a major pathway for repair in mammalian genomes (9). However, in contrast with the idea that repair generally minimizes genetic change, it has been reported that NHEJ is sometimes associated with the insertion of novel DNA fragments into DSBs in yeast (15–18), plant somatic cells (19,20) and mammalian cells (6,8,12,21). Such DNA capture events may play important and ongoing roles in the evolution of eukaryotic genomes. Previously, we observed that 8% of recovered genomic DSB repair events in mouse fibroblasts were accompanied by the insertion of DNA sequences (12). Strikingly, nearly half of the DNA capture events involved a specific fragment derived from the I-SceI expression plasmid that had been electroporated into the cells to induce a genomic DSB. Several other DNA capture events involved retrotransposable elements. From previous work it was not clear whether the capability of being captured at a DSB is a property displayed by a limited number of DNA motifs or if, instead, any sufficiently abundant extrachromosomal DNA may be captured efficiently at a DSB. In this work we addressed this question by transfecting X174 DNA into mouse fibroblasts in which a genomic DSB was simultaneously induced with I-SceI. We report that a variety of fragments of the X174 genome were captured at the DSB with high efficiency, suggesting that any extrachromosomal DNA molecule may be recruited for repair of a DSB in a mammalian genome.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Mouse Ltk– cells and derivatives were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 0.1 mM minimal non-essential amino acids (Gibco) and 50 µg gentamicin sulfate/ml. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
DSB repair substrate and cell lines
The construction of plasmid pYL1 (Fig. 1A) was described previously (12). Plasmid pYL1 contains a herpes simplex virus type 1 tk gene on a 2.5 kb BamHI fragment. Inserted into the SstI site at position 963 of the tk gene, numbering according to Wagner et al. (22), is a 24 bp oligonucleotide containing the 18 bp recognition sequence for I-SceI (Fig. 1B). Insertion of the 24 bp sequence does not disrupt tk function. Mouse Ltk– cells were electroporated with pYL1 DNA and stable tk-positive transformants containing pYL1 and resistant to medium containing hypoxanthine/aminopterin/thymidine (HAT; Sigma) were selected as previously described (12). Based on Southern blotting analysis (data not shown), we determined that cell lines 2 and 4 each contain a single integrated copy of pYL1. We used these two cell lines in the current study as well as in our previous study (12).
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DSB-induction and recovery of tk-deficient mutants
Plasmid pCMV-I-SceI, kindly provided by Maria Jasin (Sloan Kettering), contains a gene encoding I-SceI endonuclease under the control of the cytomegalovirus promoter and expressible in mouse cells (23). Prior to DSB-induction, cell lines containing pYL1 were grown in HAT medium for 4 days to kill cells that had spontaneously mutated to tk–. HAT selection was removed, and cells were allowed to continue to grow in DMEM for 5 additional days before DSB-induction. To induce a DSB, cells (5 x 106) were suspended in 800 µl phosphate buffered saline (PBS) containing 20 µg pCMV-I-SceI alone or in conjunction with either 5 µg of a HaeIII digest of
X174 DNA (New England Biolabs) or 5 µg of supercoiled X174 RFI DNA (New England Biolabs). Cells were electroporated in a Bio-Rad Gene Pulser set to 1000 V and 25 µF. For mock experiments, cells were electroporated in PBS alone. After electroporation, cells were plated at a density of 105 cells per 150 cm2 flask. The cells were grown in DMEM for 6 days under no selection and then re-fed with medium supplemented with 5 µg trifluorothymidine (TFT) per ml to select for tk-deficient clones as described previously (12). Cells were re-fed with TFT-containing medium every 3 days for 1 week until TFT-resistant (TFTR) colonies were picked. Individual TFTR clones were propagated further and genomic DNA was prepared from clones as described (9).
Southern blotting analysis
DNA was transferred to nitrocellulose filters and hybridized with a 32P-labeled probe specific for the herpes simplex virus type 1 tk gene or for X174 genomic DNA as previously described (24). The X174-specific probe was made from a HaeIII digest of X174 genomic DNA (New England Biolabs).
PCR and DNA sequencing analysis
A segment of the tk gene spanning the position of the I-SceI-induced DSB was amplified from genomic DNA isolated from TFTR clones using the primers AL1 (5'-CCAGCGTCTTGTCATTGGCG-3') and AW-22 (5'-CGGTGGGGTATCGACAGAGT-3'). Primer AL1 is nucleotides 308–327 of the coding strand of the herpes simplex virus type 1 tk gene, while primer AW-22 is nucleotides 1786–1767 of the non-coding strand of the tk gene. PCR reactions were accomplished using Ready-To-Go PCR beads (Pharmacia) using a ‘touchdown’ PCR protocol. The annealing temperature initially was set to 72°C and was progressively decreased in steps of 2°C down to 62°C with two cycles at each temperature. An additional 20 cycles were carried out at an annealing temperature of 60°C. PCR products were routinely processed with a Presequencing Kit (Amersham) and then sequenced using a Sequenase Kit Version 2.0 (Amersham) using primer AW-28 (5'-TCTACACCACACAACACCGCC-3'), nucleotides 812–831 of the tk coding strand, and primer AW43 (5'-GGCAAGGTCGGCGGGATGAG-3'), nucleotides 1111–1092 of the non-coding strand of the tk gene. Some sequencing was accomplished by automated sequencing using a Licor 4000L at the DNA Sequencing and Synthesis Core Facility in the Department of Biological Sciences at the University of South Carolina.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental system
To study DSB repair-associated mutations in a mammalian genome, mouse Ltk– fibroblast cell lines were isolated that contain a stably integrated copy of the DNA substrate pYL1 (Fig. 1A). Plasmid pYL1 contains a functional herpes simplex virus tk gene with an inserted 24 bp oligonucleotide containing the 18 bp recognition site for yeast endonuclease I-SceI (Fig. 1B). To induce a genomic DSB in the integrated tk gene, cells were electroporated with plasmid pCMV-I-SceI, which expresses endonuclease I-SceI in mammalian cells. Due to the large size of the I-SceI recognition site (18 bp), it is likely that only a single genomic DSB is induced by expression of I-SceI. Linearized or supercoiled forms of
X174 DNA were electroporated along with pCMV-I-SceI in experiments designed to assess whether transfected DNA sequences may be efficiently captured at a genomic DSB. Mutagenic DSB repair events were recovered by selecting for cells that lost tk function and thus gained resistance to the thymidine analog TFT. Selection for loss-of-tk-function should in principle allow recovery of virtually any type of non-lethal DSB-induced mutation, including capture of DNA sequences.
Recovery of DSB-induced TFTR clones
Two cell lines (2 and 4) were isolated that contain one copy of construct pYL1. Electroporation of cells with pCMV-I-SceI alone or in conjunction with a HaeIII digest of X174 DNA brought about a significant increase in the recovery of TFTR clones compared with mock electroporations (Table 1).
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tk sequences were PCR-amplified from genomic DNA isolated from a total of 132 TFTR clones (64 TFTR clones derived from cell line 2, 68 TFTR clones derived from cell line 4) following electroporation with pCMV-I-SceI plus the HaeIII digest of
X174 DNA. Primers were used that were positioned 1.5 kb apart from one another and flanked the I-SceI recognition site originally present in the integrated tk gene (see Fig. 1A). As expected, a PCR product of 1.5 kb was generated when parental genomic DNA was used as template (Fig. 2, lane 12). Among TFTR clones, 109 clones (82%) produced PCR products of 1.5 kb, suggestive of small insertions or deletions at the DSB site as seen previously (12). These clones were not analyzed further. Eighteen TFTR clones (13.6%) produced PCR products that were visibly larger than 1.5 kb (for example, Fig. 2, lanes 2 and 10). To be scored as ‘large’ in our assay, a PCR product had to be 1.6 kb or larger. The large PCR products were suggestive of capture of DNA sequences at the I-SceI-induced DSB. In our earlier work (12), a similar percentage of DSB repair events recovered from the very same two cell lines was associated with DNA capture. The large PCR products were sequenced and are described in further detail below. Five of the clones (3.8%) did not give PCR products. These clones, which either lost the entire integrated substrate or suffered a gross chromosomal rearrangement, were not studied further.
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Capture of transfected
X174 DNA at a genomic DSBSequence analyses of the 18 large PCR products revealed that DNA fragments ranging from 180 to 1622 bp had indeed been captured at the I-SceI-induced DSB (Table 2). In most of these clones, DNA capture was accompanied by deletions of 1–40 bp from one or both DNA termini at the DSB site. In several clones, capture of DNA was accompanied with the addition of a few nucleotides that may be derived from the single-strand ATAA overhang produced by I-SceI cleavage (Fig. 1B). No clone had captured a fragment from pCMV-I-SceI. This latter observation was notable because in our previous work involving electroporation of pCMV-I-SceI alone, 10 out of 21 DNA capture events involved a fragment of pCMV-I-SceI (12). Fourteen clones had captured one or more fragments of
X174 DNA (Table 2). Twelve clones captured one continuous X174 DNA sequence. In 11 of these cases, internal segments of the HaeIII fragments were captured with the sites of joining between X174 and genomic DNA positioned 6–867 bp away from the original ends of HaeIII fragments of X174. Only one clone (2-D1) captured an original X174 HaeIII fragment in its entirety. Clones 4-B11 and 4-F7 each contained portions of three distinct HaeIII fragments of X174 DNA. The captured X174 fragments are illustrated schematically in Figure 3. The inserts were derived from various regions of the X174 genome and there was no evidence for preferential capture of any particular sequence from the X174 genome.
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Capture of transfected DNA at a DSB is not accompanied by random integration
It was conceivable that the cells that captured DNA at the DSB may have been particularly proficient at incorporating exogenous DNA at all genomic loci. We were thus curious to learn whether capture of
X174 DNA at the I-SceI-induced DSB was associated with additional random integration of X174 DNA at one or more additional sites in the genome. DNA from clones that had captured X174 DNA were digested with BamHI and analyzed on a Southern blot using a X174-specfic probe (Fig. 4). As there are no BamHI sites in X174 DNA or the tk gene, we expected each clone to display a single hybridizing band if there were no sites of integration of X174 other than the I-SceI-induced DSB. We expected the size of the hybridizing fragment containing captured X174 DNA to be roughly equal to the size of the BamHI fragment from pYL1 (2.5 kb) plus the length of the captured X174 DNA. As shown in Figure 4, the X174 probe hybridized to a single band in all clones analyzed, indicating that in cells in which X174 DNA was captured at the DSB there were no additional sites of random integration of X174 DNA. Our observations indicate that transfected DNA specifically integrated at the genomic DSB site.
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DNA capture involves joining of DNA molecules preferentially at sites of microhomology
Although we transfected cells with a HaeIII digest of
X174 DNA, internal segments of the HaeIII fragments rather than the original fragments themselves were predominantly captured at the DSB. As indicated in Figure 5, in all clones (with the exception of clone 2-D1) genomic sequences were joined to X174 DNA at sites located 6–867 bp from the original termini of the HaeIII fragments. Sequence analysis revealed microhomologies of 1–4 bp at most sites of DNA joining (Fig. 5). Considering all the clones that had captured X174 DNA, there are a total of 32 novel genomic/X174 or X174/X174 DNA junctions. Illustrated in Figure 6 are the numbers of junctions displaying various lengths of microhomology compared with the distribution we would have expected to recover had DNA joining occurred at random sites. From this analysis, we conclude that DNA capture involves the joining of DNA molecules preferentially at sites of microhomology.
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Transfected linear DNA molecules are captured more efficiently than are supercoiled molecules
From the results described above, it was clear that HaeIII-digested
X174 DNA was captured at the I-SceI-induced genomic DSB more frequently than the cotransfected pCMV-I-SceI DNA. One possible explanation for this observation was that linear DNA is more efficiently captured than circular, supercoiled DNA (the disposition of the electroporated pCMV-I-SceI DNA). To address this question, we cotransfected cell lines 2 and 4 with 20 µg pCMV-I-SceI DNA (supercoiled) plus 5 µg supercoiled X174 RF I DNA. Out of a total of 69 TFTR colonies analyzed by PCR as above, six clones appeared to have captured a DNA sequence at the DSB based on the size of PCR products (data not shown). The PCR products of these six clones were further analyzed by Southern blotting using either pCMV-I-SceI or X174 DNA as a probe. Two out of six PCR products hybridized to pCMV-I-SceI and none hybridized to X174 DNA (data not shown), indicating that two clones had captured pCMV-I-SceI DNA and no clone had captured X174 DNA. This contrasted sharply with the observation that 14 out of 18 capture events involved X174 DNA when the X174 DNA was electroporated as a set of HaeIII fragments. This result suggests that linear DNA fragments are more readily captured at a genomic DSB than are circular, supercoiled DNA molecules.
Capture of other sequences at a genomic DSB
In experiments in which cell lines 2 and 4 were transfected with pCMV-I-SceI plus a HaeIII digest of X174 DNA, four of 18 TFTR clones had captured DNA sequences from sources other than X174 DNA (Table 2). Clone 2-A9 had an insert of 515 bp of continuous DNA sequence from the Escherichia coli genome, which presumably was electroporated as a contaminant along with the X174 or pCMV-I-SceI DNA. Clone 4-B5 captured a DNA fragment of 180 bp from an unidentified source. Clones 2-C7 and 2-B1 each captured a long terminal repeat (LTR) of the intracisternal A particle (an endogenous, moderately repetitive retrovirus-like sequence). The latter two clones are notable in that we previously reported several other instances of capture of similar LTR sequences (12). In addition to the LTR sequence, clone 2-B1 captured a sequence of unidentified origin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously reported (12) that capture of DNA sequences at a DSB accompanies nearly 10% of recovered DSB repair events in mouse fibroblasts. Although our previous work was consistent with the notion that any abundant extrachromosomal sequence may be captured [as had been suggested for DNA capture in yeast (18)], our work was equally consistent with the possibility that only certain specific DNA sequences or motifs are readily captured.
To gain further insight into this latter issue, in the current work we cotransfected X174 DNA along with pCMV-I-SceI to learn if X174 sequences may be readily captured at the DSB. We used a HaeIII digest of X174 DNA as a model for non-specific, low-complexity DNA having no homology to the DSB locus. Similar to previous findings for cell lines 2 and 4 (12), 13.6% of DSB-induced TFTR clones recovered from cell lines 2 and 4 in the current work captured DNA at the DSB. However, in the current work 14 out of 18 DNA capture events involved capture of one or more segments of X174 and none involved pCMV-I-SceI (Table 2). Further, there was no preference for capture of any particular region of the X174 genome. These results suggest that virtually any abundant extrachromosomal DNA sequence may be captured at a genomic DSB.
It is likely that the high frequency of capture of pCMV-I-SceI in our previous work (12) was due to the high relative abundance of pCMV-I-SceI sequences. In the current work, the absolute intracellular abundance of pCMV-I-SceI was presumably the same as in previous work as the same amount of pCMV-I-SceI DNA was electroporated. Indeed, pCMV-I-SceI was actually transfected in a 4-fold excess over X174 fragments in terms of mass (20 versus 5 µg). The reduction in pCMV-I-SceI capture concomitant with the high frequency of X174 DNA capture suggests that the X174 DNA fragments ‘out-competed’ pCMV-I-SceI for interactions with the genomic DSB. However, in transfections with supercoiled X174 RFI DNA, two out of six DNA capture events involved pCMV-I- SceI and none involved X174. These results indicated that linearized X174 DNA is more readily captured than supercoiled X174 DNA. From our experiments we cannot determine whether linear DNA molecules are intrinsically more prone to being captured or if linear molecules are more efficiently transfected into the nucleus during electroporation.
Although we infer from our results reported here that any abundant extrachromosomal sequence may be captured at a genomic DSB, our previous work (12) did indicate that a specific fragment of pCMV-I-SceI spanning the SV40 and ColE1 origins was particularly prone to capture. Our collective observations suggest that the origin sequences on pCMV-I-SceI (non-functional in mouse Ltk– cells) are either hotspots for DNA breakage or hotspots for invasion by DNA termini (see below). We rule out the possibility that the origins on pCMV-I-SceI serve as special sequences needed to deliver extrachromosomal DNA to the genomic DSB as the efficient capture of X174 fragments reported here demonstrates that, in general, transfected DNA is efficiently delivered to a DSB.
We can imagine two mechanisms for the capture of transfected DNA into a genomic DSB. DNA may be joined to both DSB termini by NHEJ. Alternatively, DNA may be ‘copied’ into the genomic DSB by a break-induced replication (BIR) process (reviewed in 25) in which one or both 3' DNA ends from the genomic DSB invades a transfected molecule (likely at a site of microhomology) and primes DNA synthesis using the transfected molecule as a template. In the case of a one-ended invasion, the other end of the genomic DSB would join with the newly copied DNA via NHEJ. The finding of microhomologies at sites of joining between captured DNA and genomic sequences (Figs 5 and 6) is consistent with either NHEJ or BIR. The fact that most of the X174 inserts are internal fragments rather than the original HaeIII fragments argues that if NHEJ is often involved in capture then substantial degradation of fragment ends typically occurs prior to most capture events. If BIR is an operative mechanism for capture, invasion of genomic DNA termini at internal sites of microhomology may occur with little or no degradation of the HaeIII fragment ends. We cannot distinguish between invasion at internal sites versus exonucleolytic digestion of fragment ends, although earlier work by others (2) suggests that random integration into a mammalian genome frequently occurs with the loss of very few nucleotides from the termini of transfected DNA fragments. We also note that Merrihew et al. (26) have presented evidence for a ‘copy-join’ mechanism for random integration into a mammalian genome. In this scenario, integration of transfected DNA is preceded by one or more rounds of invasion of a terminus of the transfected DNA molecule into genomic sites of microhomology. During these events, the invading terminus serves as a primer for DNA synthesis resulting in the addition of genomic sequences to the end of the transfected molecule. The transfected molecule may thus ‘pick up’ segments of genomic sequence from several loci prior to integrating. In a somewhat analogous fashion, we propose that DNA termini from a broken chromosome may invade transfected DNA molecules at sites of microhomology, allowing the genome to ‘pick up’ segments of transfected sequences.
Our work makes it evident that DNA transfected into a mammalian cell will preferentially integrate at a genomic DSB. In our hands, stable integration of a selectable marker typically occurs at a frequency of less than one in 104 electroporated mouse Ltk– cells (data not shown). X174 DNA was captured in about one in 103 electroporated cells (Table 1). Considering the small ‘target’ size of the I-SceI-induced DSB in the context of the entire genome, it is unequivocal that the transfected X174 DNA preferentially integrated at the DSB. It would appear that there exists a mechanism that results in the efficient delivery of transfected DNA to the site of a genomic DSB. We note previous reports of preferential integration of transfected DNA at unstable mammalian loci (25) and at an induced fragile site (27), consistent with the proposition that DNA efficiently integrates at sites of genomic breakage. Selective integration of transfected DNA at a genomic DSB may have future application in methodologies for targeted genome alterations.
Our work suggests that mammalian cells exhibit one or more robust DSB repair pathways that can apparently lead to patching of genomic DSBs by virtually any DNA molecule that happens to be available. To maintain genomic integrity, cells undoubtedly have regulatory mechanisms for selecting the DNA sequences with which the ends of a broken chromosome will interact. How cells may control DSB repair to help avoid promiscuous exchanges is a topic requiring further investigation.
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ACKNOWLEDGEMENTS |
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We thank Dr Barbara Criscuolo Waldman for helpful comments on the manuscript. This work was supported by grants from the American Cancer Society (NP-949) and the National Institute of General Medical Sciences (GM47110) to A.S.W.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 803 777 8405; Fax: +1 803 777 4002; Email: awaldman{at}sc.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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X. Yu and A. Gabriel Ku-Dependent and Ku-Independent End-Joining Pathways Lead to Chromosomal Rearrangements During Double-Strand Break Repair in Saccharomyces cerevisiae Genetics, March 1, 2003; 163(3): 843 - 856. [Abstract] [Full Text] [PDF]
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