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J. Biol. Chem., Vol. 277, Issue 48, 46243-46247, November 29, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/48/46243    most recent M207937200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Donahue, S. L. Articles by Campbell, C. Search for Related Content PubMed PubMed Citation Articles by Donahue, S. L. Articles by Campbell, C. Social Bookmarking                      What's this?

A DNA Double Strand Break Repair Defect in Fanconi Anemia Fibroblasts^*

Sarah L. Donahue and Colin Campbell

From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Received for publication, August 5, 2002, and in revised form, September 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fanconi anemia (FA) is a heterogeneous autosomal recessive disease characterized by congenital abnormalities, pancytopenia, and an increased incidence of cancer. Cells cultured from FA patients display elevated spontaneous chromosomal breaks and deletions and are hypersensitive to bifunctional cross-linking agents. Thus, it has been hypothesized that FA is a DNA repair disorder. We analyzed plasmid end-joining in intact diploid fibroblast cells derived from FA patients. FA fibroblasts from complementation groups A, C, D2, and G rejoined linearized plasmids with a significantly decreased efficiency compared with non-FA fibroblasts. Retrovirus-mediated expression of the respective FA cDNAs in FA cells restored their end-joining efficiency to wild type levels. Human FA fibroblasts and fibroblasts from FA rodent models were also significantly more sensitive to restriction enzyme-induced chromosomal DNA double strand breaks than were their retrovirally corrected counterparts. Taken together, these data show that FA fibroblasts have a deficiency in both extra-chromosomal and chromosomal DNA double strand break repair, a defect that could provide an attractive explanation for some of the pathologies associated with FA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fanconi anemia (FA)¹ is a fatal inherited autosomal recessive disease characterized by progressive bone marrow failure and a significant predisposition toward malignancies, particularly acute myelogenous leukemia (1, 2). Somatic cell hybridization studies have shown that abnormalities in multiple genes result in FA (3-5). To date, at least eight distinct complementation groups have been identified (A, B, C, D1, D2, E, F, and G), and all of the known FA genes have been cloned (6-12). With the exception of FANCB, and FANCD1, none of the FA genes contains sequence motifs of known function (12), and only FANCD2 has been found to have a homolog in lower eukaryotic organisms (5). Some of the FA genes encode proteins that interact to form a complex in the nucleus. This complex is disrupted in cell lines from complementation groups A, C, E, F, and G (13, 14). Yet despite these findings, the exact biological functions of the FA proteins have not yet been determined and the molecular mechanism(s) responsible for FA have remained obscure (15).

Interestingly, although the molecular defect responsible for FA is unclear, it has been well documented that cells from FA patients display elevated levels of spontaneous chromosomal breaks and deletions and have an increased sensitivity to the cytotoxic and clastogenic effects of DNA cross-linking agents (16-19). Patient-derived FA lymphoblasts have been shown to have significantly decreased plasmid-rejoining fidelity compared with normal lymphoblasts (20, 21). Additionally, nuclear extracts from patient-derived FA fibroblasts have substantially decreased plasmid-rejoining activity compared with extracts from normal fibroblasts (22). These cellular features along with the high susceptibility of FA patients to cancers have lead to the hypothesis that this disorder results from defective DNA repair. However, the absence of recognizable DNA binding sequence motifs in FA genes and the lack of evidence showing direct interaction of FA gene products with DNA cast doubt on the idea that FA proteins directly participate in DNA repair. Recent results demonstrating a connection between the BRCA1 and BRCA2 tumor suppressor and FA proteins suggest that FA proteins may play an essential role in regulating the cellular response to DNA damage (12, 15, 23-25).

We examined both extra-chromosomal and chromosomal DNA repair in intact FA fibroblasts and retrovirally corrected FA fibroblasts from multiple complementation groups and animal models. We found that fibroblasts derived from FA patients of complementation groups A, C, D2, and G were significantly deficient in the repair of both plasmid and chromosome DNA double strand breaks and that this deficiency was corrected by the expression of the respective FA cDNAs in these cells. Furthermore, a similar defect in DNA double strand break repair was seen in cells derived from two rodent models of FA and in wild type cells expressing a dominant negative FANCC allele.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Cells were maintained in a humidified 5% CO₂-containing atmosphere at 37 °C. All FA cell lines were obtained from the Oregon Health Sciences University unless otherwise noted. HT1080 are immortalized human sarcoma-transformed fibroblast cells. MCF-7 (ATCC Cell Repository) and MA148 are human-immortalized non-FA epithelial cells. CCL75.1, GM637, GM638, GM847, and GM10603 (NIGMS, National Institutes of Health Human Genetic Cell Repository) are immortalized human non-FA fibroblasts. PD.715.F and PD.792.F are normal human-diploid fibroblasts. PD.220i, PD.20i, and PD.20hygro (referred to as A', D', and D") are human immortalized FA fibroblasts of complementation groups A, D2, and D2, respectively. PD.20hygro:RV (referred to as D'-corrected) are PD.20hygro cells that have been infected with the retrovirus that expresses the FANCD2 cDNA. PD.720.F, 551-FAA, and PD.352.F (referred to as A", C, and G) are human diploid FA fibroblasts from complementation groups A, C, and G, respectively. 720-Retro, 551-FAC, and 352-FAG (referred to as A"-corrected, C-corrected, and G-corrected) are human primary FA fibroblasts that have been infected with retroviruses that express the FANCA, FANCC, and FANCG cDNAs, respectively. Murine MPF60T, MPF61T, and MPF62T cells are embryonic fibroblasts derived from mice homozygous for Fancc^exon9, heterozygous for Fancc^exon9, and homozygous wild type, respectively (26). Genotypes of these cells were verified by PCR analysis of genomic DNA. Chinese hamster ovary cells AA8 and UV40 (Xrcc9 mutants) were kindly provided by Dr. Larry H. Thompson (Lawrence Livermore National Laboratory, Livermore, CA) (27). The patient-derived wild type and L554P FANCC alleles were kindly provided by Dr. Maureen E. Hoatlin (Oregon Health Sciences University and Portland Veterans Affairs Medical Center, Portland, OR) (28).

DNA End-Joining-- Experiments were carried out as described previously (20) with the following exceptions. Plasmid pSV2Neo was used as the substrate DNA. It was treated with the endonucleases EcoRI to produce cohesive DNA ends and SmaI to produce blunt ends. Linear plasmid was gel-purified and quantitated. Five micrograms of plasmid pRSVEdl884 that encodes the SV40 large T-antigen was co-transfected into cells along with 1.25 µg of pSV2Neo. Plasmid DNA recovered after DpnI digestion was electroporated into DH10B-electrocompetent bacteria.

Restriction Enzyme Electroporation-- Approximately 10⁴ fibroblasts were collected and resuspended in 200 µl of serum-free media along with restriction enzymes PvuII or HinfI (Invitrogen) diluted in their respective storage buffers to concentrations of 0, 10, 20, 40, and 60 units/50 µl. For heat inactivation, enzyme was diluted to the appropriate concentration and incubated for 18 h at 70 °C. The cell-enzyme mixture was electroporated with a BTX ECM 630 electroporator (Genetronic Inc., San Diego, CA) at an electrical field strength of 0.75 kV/cm and a capacitance of 960 microfarads. Cytotoxicity was examined using the sulforhodamine B (SRB) assay (29) or clonogenic assay. For the SRB assay, electroporated cells were plated into 24-well dishes followed by incubation for 24 h. Percent cell survival was determined by comparing the optical density at 564 nm for cells electroporated with 0 units of enzyme to the optical density for cells electroporated with 10-60 units of enzyme. For the clonogenic assay, electroporated cells were plated into 100-mm dishes and allowed to grow and form colonies for 2-3 weeks. The number of colonies from electroporation with 0 units of enzyme was compared with the number of colonies obtained from electroporation with 10-60 units of enzyme.

Restriction Enzyme Poration Mediated by Streptolysin O-- PvuII was introduced into cells by poration with streptolysin O as described previously (30). Immediately afterward, cells were plated into 24-well dishes and incubated for 24 h, and percent survival was determined by the SRB assay as previously stated.

Green Fluorescent Protein Electroporation-- Twenty-five micrograms of recombinant green fluorescent protein (Clontech, Palo Alto, CA) was electroporated into cells. After 6 h, cells were examined by confocal microscopy. The uptake of the green fluorescent protein was determined by comparing the number of cells fluorescing at 522 nm (excitation 488 nm) to the number of cells observed in the bright field.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid End-Joining in Intact Wild Type and FA Fibroblasts-- DNA end-joining was examined within intact fibroblast cells by electroporating linear plasmid DNA molecules into fibroblasts. Rejoined plasmids were recovered from fibroblasts and analyzed with a bacterial reporter system. The frequency with which the linearized DNA was rejoined was determined by comparing the number of bacterial colonies obtained when fibroblasts were electroporated with linear plasmid DNA to the number obtained when fibroblasts were electroporated in a parallel experiment with circular plasmid DNA (20). An examination of a number of wild type cell lines revealed that plasmids with cohesive DNA ends were rejoined with ~28% efficiency and that there were no significant differences among any of the cells examined (Table I). In contrast, the rejoining efficiency of the same plasmid substrate was only ~4% in fibroblasts derived from FA patients from complementation groups A, C, D2, and G (Table I). Additional experiments using blunt-ended plasmid DNA revealed similar results. FA fibroblasts of complementation groups A, C, D2, and G had rejoining frequencies with blunt-ended substrate of 3.0, 5.1, 3.1, and 4.4%, respectively, whereas the efficiency of rejoining of these substrates in non-FA cells was ~30%. To verify that all rejoined plasmids recovered by the bacterial reporter system were derived exclusively from substrates processed within human cells, both cohesive-ended and blunt-ended linearized plasmid substrate was electroporated directly into bacteria. As expected, no bacterial colonies were obtained from this control experiment (data not shown).

Interestingly, we had previously found that nuclear extracts prepared from fibroblasts derived from eight unrelated normal human donors rejoined linear plasmids with an average efficiency of 26%, whereas extracts from fibroblasts derived from three unrelated FA patients rejoined these plasmids with efficiencies ranging from 4 to 12% (22). An analysis revealed that the majority of products from both the cell-free and intracellular rejoining reactions were precisely rejoined, whereas a substantial minority had suffered small deletions that spanned direct sequence repeats.

Plasmid rejoining was also examined in FA cell strains that had been "corrected" through infection by retroviruses encoding the corresponding FA cDNA. It has previously been demonstrated that such correction restored resistance to cross-linking agents in these FA cell strains (6, 8-11). As Table I reveals, in all four cases, the retrovirally corrected FA cells rejoined cohesive-ended DNA with frequencies similar to that seen in non-FA cells. Additionally, the end-joining efficiency of blunt-ended DNA substrate was ~30% in these cells. As was expected, infection of an FANCC cell strain with a retrovirus encoding an FANCA cDNA had no effect on plasmid-rejoining efficiency (data not shown).

Hypersensitivity of FA Fibroblasts to Restriction Enzyme-induced Cell Death-- Cells cultured from FA patients display an increased level of chromosomal abnormalities including spontaneous breaks and deletions (1). This along with the severe plasmid-rejoining defect seen in FA fibroblasts prompted us to hypothesize that FA fibroblasts would be sensitive to induced chromosomal DNA double strand breaks. To test this hypothesis, intact FANCD2 cells and their retrovirus-corrected counterparts were electroporated with restriction endonucleases. This treatment has been shown to create chromosomal DNA double strand breaks, ultimately resulting in cell death (31-35). Fibroblasts were electroporated with the restriction enzyme PvuII, which creates blunt-ended chromosomal double strand breaks. Table II shows that neither 10 nor 20 units of PvuII had a significant effect on cell survival in several non-FA fibroblast cell lines and strains. Conversely, a similar treatment of FA fibroblasts from complementation groups A, C, D2, and G with PvuII resulted in significantly less cell survival (Table II). Retroviral correction with the corresponding FA cDNAs, however, restored restriction enzyme resistance in FA cells. Heat inactivation of PvuII prior to electroporation abolished their ability to induce cell death (data not shown). Similar results were obtained when the restriction enzyme HinfI, which creates cohesive-ended chromosomal double strand breaks, was introduced into cells (data not shown).

Cell viability following these restriction enzyme treatments was determined using the SRB assay (29). To ensure that this assay accurately measured cytotoxicity, analogous experiments were analyzed using clonogenic assays. A similar decrease in survival was seen in FANCD2 fibroblasts compared with retrovirally corrected FANCD2 cells when survival was determined through colony formation (data not shown). To rule out the possibility that the decreased cell survival of FA fibroblasts was caused by the deleterious affects of electroporation, streptolysin O treatment was used to introduce PvuII into cells (30). A decrease in cell survival similar to that observed following enzyme electroporation was seen after streptolysin O-mediated poration of PvuII (data not shown). Additionally, the SRB and clonogenic assays both showed that cell death attributed to poration alone was comparable in FA and non-FA cells following electroporation and streptolysin O-mediated poration, resulting in ~40-60% killing in all cells examined.

As Table II reveals that although FA fibroblast cells from complementation groups A, C, D2, and G displayed increased restriction enzyme-induced cell death, their retrovirus-corrected counterparts had sensitivities similar to non-FA cells. To ensure that the reduced sensitivity of retrovirus-corrected FA cells to restriction enzymes was not because of a difference in transfection efficiency of porated protein, we examined the extent to which they and their unmodified counterparts took up recombinant green fluorescent protein. We found that 69.8 ± 2.4% of uncorrected FANCD2 cells accumulated significant levels of green fluorescent protein following electroporation compared with 71.2 ± 1.1% of the corrected cells. Since both FA and retrovirus-corrected FA fibroblasts are able to take up similar amounts of electroporated protein, the observed sensitivity to restriction enzyme-induced cell death is not because of increased restriction enzyme uptake. It is noteworthy that the percent of cells taking up electroporated green fluorescent protein is roughly similar to the number of FA cells killed after restriction enzyme poration. This result suggests that all of the FA cells that take up restriction enzymes are killed, although the non-FA cells and corrected FA cells that take up restriction enzymes are relatively resistant to their cytotoxic affects.

DNA End-Joining and Chromosomal Double Strand Break Sensitivity in HT1080 Cells Expressing a Dominant Negative FANCC Allele-- The finding that overexpression of a patient-derived FANCC allele (L554P) rendered normal cells sensitive to the cross-linking agent diepoxybutane (28) prompted us to ask whether overexpression of this dominant negative allele in HT1080 cells would also render them deficient in plasmid rejoining and hypersensitive to restriction enzyme-induced cell death. As Fig. 1A indicates, transgenic HT1080 fibroblasts expressing the L554P FANCC allele were hypersensitive to diepoxybutane. Furthermore, these cells rejoined both cohesive-ended and blunt-ended linearized plasmids with significantly diminished efficiency (Fig. 1B). HT1080 cells expressing L554P were also hypersensitive to restriction enzyme-induced cell death following electroporation of PvuII (Fig. 1C). The decreases in plasmid end-joining and cell survival following restriction enzyme treatment were of similar magnitude to those observed in the patient-derived FA fibroblasts of complementation groups A, C, D2, and G. Overexpression of the wild type FANCC allele in HT1080 cells had no effect on any of the parameters examined (Fig. 1A-C).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   HT1080 fibroblasts expressing the patient-derived mutant FANCC allele L554P resemble FA cells. A phenotypic analysis was performed on HT1080 cells (black bars) as well as on HT1080 cells that overexpressed a wild type FANCC allele (shaded bars) and HT1080 cells that overexpressed the L554P FANCC allele (white bars). Data represent the mean of three experiments; error bars depict the mean ± S.E. *, p < 0.005. A, sensitivity to diepoxybutane. B, plasmid-rejoining efficiency. C, sensitivity to restriction enzyme-induced DNA double strand breaks.

Restriction Enzyme-induced Cell Death in Fibroblasts from Two Rodent Models of FA-- We next examined whether the hypersensitivity to restriction enzyme-induced cell death seen in human FA fibroblasts was also observed in two rodent models of FA. Embryonic fibroblasts derived from mice homozygous for a targeted deletion of exon 9 of the murine FA complementation group C gene (Fancc^exon9) display chromosomal breaks and are hypersensitive to DNA cross-linking agents in a manner similar to human FA cell strains (26). As Fig. 2A indicates, these murine fibroblasts were also hypersensitive to restriction enzyme-induced cell death, whereas wild type murine embryo fibroblasts and murine embryo fibroblasts heterozygous for the deleted Fancc allele were resistant. Likewise, Chinese hamster ovary-derived UV40 cells (27), which fail to produce functional Xrcc9 protein (the hamster homolog of human FANCG protein) and have also been shown to be hypersensitive to DNA cross-linking agents (36), were significantly more sensitive to restriction enzyme-induced cell death than were cells of the wild type parental cell line AA8 (Fig. 2B). These data indicate that the FA-like rodent cells are as sensitive to restriction enzyme-induced killing as human FA fibroblasts.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Mouse fibroblasts deficient in Fancc are sensitive to restriction enzyme-induced DNA double strand breaks. Data represent the mean of three experiments; error bars depict the mean ± S.E., *, p < 0.005. A, restriction enzyme-induced cell death was measured in mouse embryonic fibroblasts homozygous for an Fancc exon 9 deletion allele (), normal mouse fibroblasts (), and mouse fibroblasts heterozygous for the Fancc exon 9 deletion allele (). B, restriction enzyme-induced cell death was measured in Xrcc9-deficient UV40 cells () and the UV40 parental cell line AA8 ().

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The data presented herein support the conclusion that FA fibroblasts have a dramatically reduced ability to rejoin double strand breaks in both introduced plasmids as well as within their chromosomes. These defects were observed in diploid fibroblasts from patients whose cells belong to a number of different FA complementation groups. In all cases tested, the re-introduction of the deficient FA gene into these cells eliminated the aberrant phenotype. An inability to repair restriction enzyme-induced chromosomal double strand breaks was also observed in two different rodent models of FA as well as in a human cell culture model of FA induced by overexpression of a dominant negative allele of the FANCC gene. Taken together, these findings provide robust support for the conclusion that FA fibroblasts have a defect in cellular DNA double strand break repair.

The nature of the DNA double strand break repair defect in FA fibroblasts, however, remains obscure. It is known that mammalian cells use both recombinational and non-homologous end-joining pathways to repair DNA double strand breaks. Thus, in principle, either mechanism or conceivably both could be affected in FA fibroblasts. It is not likely that the linearized plasmid substrates utilized in our end-joining assays are repaired via a recombinational mechanism. Thus, we can conclude that FA fibroblasts have a defect in a non-recombinational DNA double strand break repair pathway. The findings that V(D)J recombination and Ku-dependent non-homologous end-joining are not affected in FA cells (20-22, 37) indicate that this observed defect does not involve these pathways. Instead, it appears that the deficiency resides in another currently uncharacterized non-recombinational repair pathway.

It is tempting to speculate that the hypersensitivity of FA fibroblasts to restriction enzyme-induced chromosomal double strand breaks is a consequence of deficient non-recombinational repair of these lesions. However, a number of findings suggest that this hypersensitivity could reflect a deficiency in chromosomal homologous recombinational repair. First, the BRCA1 protein, which is required for efficient recombinational repair of chromosome double strand breaks, co-localizes to nuclear foci with the FANCD2 protein in cells following exposure to ionizing radiation (24). FANCD2 has also been shown to be phosphorylated in an ATM-dependent manner following induced DNA damage (25). Second, the FANCB and FANCD1 genes are apparently identical to BRCA2, a gene also required for efficient recombinational repair of chromosome double strand breaks (12, 15, 38). Third, both the BRCA1 and BRCA2 proteins interact with the mammalian RecA homolog Rad51 (39), and cells deficient in BRCA1 and/or BRCA2 have a significant defect in homologous recombination, display chromosomal instability, and are hypersensitive to DNA cross-linking agents as are FA cells (38, 40-45). Thus, it is conceivable that FA cells have a defect in recombinational repair of chromosomal DNA double strand breaks. It remains to be determined whether the elevated sensitivity of FA cells to restriction enzyme-induced cell death is a consequence of defective non-homologous end-joining, defective recombinational repair, or both. It may be possible to gain insight into this question by studying the repair of double strand breaks induced into engineered chromosomal loci by rare-cutting endonucleases such as the yeast I SceI enzyme.

An alternative explanation for the cytotoxicity observed in FA fibroblasts following introduction of restriction enzymes is that cell death may be due to improper checkpoint regulation following chromosomal damage. A recent report by Taniguchi et al. (25) shows that FANCD2 fibroblasts have radio-resistant DNA synthesis following induced DNA damage. Similarly, BRCA2/FANCD1-deficient Chinese hamster ovary cells and BRCA1-deficient cells also display radio-resistant DNA synthesis after exposure to ionizing radiation (46, 47). Thus, given the association among FA proteins and BRCA1 and BRCA2 previously outlined, these data indicate that FA cells may have an S phase checkpoint defect. Failed repair of DNA double strand breaks and an inability to regulate an essential checkpoint may result in these FA cells progressing through the cell cycle with unrepaired chromosomal lesions that would ultimately lead to cell death.

Regardless of the nature of the defect, the deficiency in DNA double strand break repair observed in FA fibroblasts may provide an attractive explanation for some of the pathologies associated with FA. Although this conclusion is derived from studies performed on fibroblast cells and may not be applicable to all cell types, evidence from lymphoblasts derived from FA patients also indicates a deficiency in DNA double strand break repair (20, 21, 48). Additionally, an examination of both fibroblasts and lymphoblasts derived from FA patients has revealed no distinct differences in sensitivities to DNA-damaging agents or chromosomal instability, the two main cellular features of FA (16, 19, 49, 50). Thus, the cancer predisposition that characterizes this disorder could result from chromosomal rearrangements is due to defective repair of chromosome double strand breaks that arise spontaneously or are created as intermediates in normal cellular processes. Likewise, just as defective repair of spontaneous DNA double strand breaks caused by oxygen is responsible for the neuronal apoptosis observed in knock-out mice lacking a functional non-homologous end-joining pathway (51), the defective DNA double strand break repair we observe in FA cells could be responsible for bone marrow failure observed in these patients. Therefore, one exciting possibility is that pharmacological approaches may be developed to activate the defective DNA double strand break repair pathway in FA cells. Such therapeutic intervention could potentially halt the inexorable loss of bone marrow stem cells that results in fatal anemia in these patients, thereby extending their life span.

	ACKNOWLEDGEMENTS

We thank Drs. Larry H. Thompson, Maureen E. Hoatlin, and Barbara Cox for kindly providing reagents used herein.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant AG16678 and the Breast Cancer Research Program Grant DAMD17-99-1-9299 from the U. S. Department of Defense.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church St., S. E., Minneapolis, MN 55455. Tel.: 612-625-8986; Fax: 612-625-8408; E-mail: campb034@tc.umn.edu.

Published, JBC Papers in Press, October 1, 2002, DOI 10.1074/jbc.M207937200

	ABBREVIATIONS

The abbreviations used are: FA, Fanconi anemia; SRB, sulforhodamine B.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/48/46243    most recent M207937200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Donahue, S. L. Articles by Campbell, C. Search for Related Content PubMed PubMed Citation Articles by Donahue, S. L.