RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous... more RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks. The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species. This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation. Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation. Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation. rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes. Conjugation of two rad51 nulls leads to an arrest early during prezygotic development (meiosis I). We di...
We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzym... more We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream AT...
Proceedings of the National Academy of Sciences, 1991
We have generated a nested series of interstitial deletions in a fragment of human X chromosome-d... more We have generated a nested series of interstitial deletions in a fragment of human X chromosome-derived DNA cloned into a yeast artificial chromosome (YAC) vector. A yeast strain carrying the YAC was transformed with a linear recombination substrate containing at one end a sequence that is uniquely represented on the YAC and at the other end a truncated long interspersed repetitive element (LINE 1, or L1). Homologous recombination between the YAC and the input DNA resulted in a nested series of interstitial deletions, the largest of which was 500 kilobases. In combination with terminal deletions that can be generated through homologous recombination, the interstitial deletions are useful for mapping and studying gene structure-function relationships.
Fanconi anemia (FA) is a fatal genetic disorder associated with pancytopenia and cancer. Cells la... more Fanconi anemia (FA) is a fatal genetic disorder associated with pancytopenia and cancer. Cells lacking functional FA genes are hypersensitive to bifunctional alkylating agents, and are deficient in DNA doublestrand break repair. Multiple genes with FA-causing mutations have been cloned, however, the molecular basis for FA remains obscure. The results presented herein indicate that a Rad50-dependent end-joining process is non-functional in diploid fibroblasts from FA patients. Introduction of anti-Rad50 antibody into normal fibroblasts sensitized them to DNA damaging agents, whereas this treatment had no effect on fibroblasts from FA patients. The DNA end-joining process deficient in FA cells also requires the Mre11, Nbs1 and DNA ligase IV proteins. These data reveal the existence of a previously uncharacterized Rad50-dependent DNA double-strand break repair pathway in mammalian somatic cells, and suggest that failure to activate this pathway is responsible, at least in part, for the defective DNA end-joining observed in FA cells.
The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence ... more The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence supports the conclusion that the nuclear DNA ligase III protein plays an essential role in both base excision repair and homologous recombination. However, the role of DNA ligase III protein in mitochondrial genome dynamics has been obscure. Human tumor-derived HT1080 cells were transfected with an antisense DNA ligase III expression vector and clones with diminished levels of DNA ligase III activity identified. Mitochondrial protein extracts prepared from these clones had decreased levels of DNA ligase III relative to extracts from cells transfected with a control vector. Analysis of these clones revealed that the DNA ligase III antisense mRNAexpressing cells had reduced mtDNA content compared to control cells. In addition, the residual mtDNA present in these cells had numerous singlestrand nicks that were not detected in mtDNA from control cells. Cells expressing antisense ligase III also had diminished capacity to restore their mtDNA to pre-irradiation levels following exposure to γ-irradiation. An antisense-mediated reduction in cellular DNA ligase IV had no effect on the copy number or integrity of mtDNA. This observaion, coupled with other evidence, suggests that DNA ligase IV is not present in the mitochondria and does not play a role in maintaining mtDNA integrity. We conclude that DNA ligase III is essential for the proper maintenance of mtDNA in cultured mammalian somatic cells.
The RAD51 gene is a eukaryotic homolog of recA, a critical component in homologous recombination ... more The RAD51 gene is a eukaryotic homolog of recA, a critical component in homologous recombination and DNA repair pathways in Escherichia coli. We have cloned the RAD51 homolog from Tetrahymena thermophila, a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single-and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m 2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.
We evaluated the effect of target site transcription on gene targeting In cultured human flbrosar... more We evaluated the effect of target site transcription on gene targeting In cultured human flbrosarcoma cells. A number of cell lines that harbored a plasmid recombination substrate within their chromosomal DNA were created. Gene targeting frequency was then measured at these different loci in the presence and absence of an agent that stimulated target site transcription. We observed that gene targeting was significantly enhanced by RNA transcription. The magnitude of transcription-stimulated gene targeting varied from 3-fold to >20-fold. No increase in gene targeting was observed, however, when transcription proceeded away from, rather than through, the recombination site. Transcription-stimulated gene targeting was also observed when single-stranded plasmid vectors complementary to either the coding or template strand were used as recombination substrates. Our results indicate that gene targeting, like other forms of DNA recombination, can be stimulated by target site transcription. The implications of our observations on current models of transcription-stimulated recombination are discussed.
Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful ... more Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins which were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI +-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.
V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2... more V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex plays a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Herein we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V (D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly 10-fold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins significantly reduced the fidelity of signal joint recombination in wild-type cells. The latter finding was somewhat unexpected because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells dramatically reduced the fidelity of signal joint recombination. These data reveal a heretofore unreported role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.
The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophospha... more The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan, is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here we show that the DNA repair protein, O 6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32 P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with guanine half mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT-a variant containing a single point mutation in the protein's active site-was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half mustards. HPLC-ESI +-MS/MS sequencing of the tryptic peptides obtained from the wild type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys 145 and Cys 150). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid and N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically.
Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to ... more Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of druginduced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O 6-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Lower numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.
The data in the present study show that DNA polymerase γ and DNA ligase III interact in mitochond... more The data in the present study show that DNA polymerase γ and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase γ was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase γ with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretat...
The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the ... more The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (∆dnl4). The Escherichia coli EcoRI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant EcoRI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and ∆dnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.
1,2,3,4-Diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate car... more 1,2,3,4-Diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate carcinogenic metabolite of the common industrial chemical and environmental carcinogen 1,3butadiene. DEB is a bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs), which are thought to play a central role in its biological activity. Previous studies with recombinant proteins have shown that the biological outcomes of DEB-induced DPCs are strongly influenced by protein identities. The present work combines affinity capture methodology with mass spectrometry-based proteomics and immunological detection to identify the proteins which form DPCs in nuclear extracts from human cervical carcinoma (HeLa) cells. We identified 39 human proteins that form covalent DPCs in the presence of DEB. DNA-protein cross-linking efficiency following treatment with 25 mM DEB was 2-12%, depending on protein identity. HPLC-ESI +-MS/MS analysis of the total proteolytic digests of cross-linked proteins revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3butanediol conjugates, suggesting that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA.
We examined unequal homologous DNA recombination between human repetitive DNA elements located on... more We examined unequal homologous DNA recombination between human repetitive DNA elements located on a yeast artificial chromosome (YAC) and transforming plasmid molecules. A plasmid vector containing an Alu element, as well as a sequence identical to a unique site on a YAC, was introduced into yeast and double recombinant clones analyzed. Recombination occurs between vector and YAC Alu elements sharing as little as 74% identity. The physical proximity of an Alu element to the unique DNA segment appears to play a significant role in determining the frequency with which that element serves as a recombination substrate. In addition, cross-over points of the recombination reaction are largely confined to the ends of the repetitive element. Since a similar distribution of crossover sites occurs during unequal homologous recombination in human germ and somatic tissue, we propose that similar enzymatic processes may be responsible for the events observed in our system and in human cells. This suggests that further examination of the enzymology of unequal homologous recombination of human DNA within yeast may yield a greater understanding of the molecular events which control this process in higher eukaryotes.
RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous... more RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks. The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species. This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation. Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation. Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation. rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes. Conjugation of two rad51 nulls leads to an arrest early during prezygotic development (meiosis I). We di...
We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzym... more We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream AT...
Proceedings of the National Academy of Sciences, 1991
We have generated a nested series of interstitial deletions in a fragment of human X chromosome-d... more We have generated a nested series of interstitial deletions in a fragment of human X chromosome-derived DNA cloned into a yeast artificial chromosome (YAC) vector. A yeast strain carrying the YAC was transformed with a linear recombination substrate containing at one end a sequence that is uniquely represented on the YAC and at the other end a truncated long interspersed repetitive element (LINE 1, or L1). Homologous recombination between the YAC and the input DNA resulted in a nested series of interstitial deletions, the largest of which was 500 kilobases. In combination with terminal deletions that can be generated through homologous recombination, the interstitial deletions are useful for mapping and studying gene structure-function relationships.
Fanconi anemia (FA) is a fatal genetic disorder associated with pancytopenia and cancer. Cells la... more Fanconi anemia (FA) is a fatal genetic disorder associated with pancytopenia and cancer. Cells lacking functional FA genes are hypersensitive to bifunctional alkylating agents, and are deficient in DNA doublestrand break repair. Multiple genes with FA-causing mutations have been cloned, however, the molecular basis for FA remains obscure. The results presented herein indicate that a Rad50-dependent end-joining process is non-functional in diploid fibroblasts from FA patients. Introduction of anti-Rad50 antibody into normal fibroblasts sensitized them to DNA damaging agents, whereas this treatment had no effect on fibroblasts from FA patients. The DNA end-joining process deficient in FA cells also requires the Mre11, Nbs1 and DNA ligase IV proteins. These data reveal the existence of a previously uncharacterized Rad50-dependent DNA double-strand break repair pathway in mammalian somatic cells, and suggest that failure to activate this pathway is responsible, at least in part, for the defective DNA end-joining observed in FA cells.
The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence ... more The human DNA ligase III gene encodes both nuclear and mitochondrial proteins. Abundant evidence supports the conclusion that the nuclear DNA ligase III protein plays an essential role in both base excision repair and homologous recombination. However, the role of DNA ligase III protein in mitochondrial genome dynamics has been obscure. Human tumor-derived HT1080 cells were transfected with an antisense DNA ligase III expression vector and clones with diminished levels of DNA ligase III activity identified. Mitochondrial protein extracts prepared from these clones had decreased levels of DNA ligase III relative to extracts from cells transfected with a control vector. Analysis of these clones revealed that the DNA ligase III antisense mRNAexpressing cells had reduced mtDNA content compared to control cells. In addition, the residual mtDNA present in these cells had numerous singlestrand nicks that were not detected in mtDNA from control cells. Cells expressing antisense ligase III also had diminished capacity to restore their mtDNA to pre-irradiation levels following exposure to γ-irradiation. An antisense-mediated reduction in cellular DNA ligase IV had no effect on the copy number or integrity of mtDNA. This observaion, coupled with other evidence, suggests that DNA ligase IV is not present in the mitochondria and does not play a role in maintaining mtDNA integrity. We conclude that DNA ligase III is essential for the proper maintenance of mtDNA in cultured mammalian somatic cells.
The RAD51 gene is a eukaryotic homolog of recA, a critical component in homologous recombination ... more The RAD51 gene is a eukaryotic homolog of recA, a critical component in homologous recombination and DNA repair pathways in Escherichia coli. We have cloned the RAD51 homolog from Tetrahymena thermophila, a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single-and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m 2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.
We evaluated the effect of target site transcription on gene targeting In cultured human flbrosar... more We evaluated the effect of target site transcription on gene targeting In cultured human flbrosarcoma cells. A number of cell lines that harbored a plasmid recombination substrate within their chromosomal DNA were created. Gene targeting frequency was then measured at these different loci in the presence and absence of an agent that stimulated target site transcription. We observed that gene targeting was significantly enhanced by RNA transcription. The magnitude of transcription-stimulated gene targeting varied from 3-fold to >20-fold. No increase in gene targeting was observed, however, when transcription proceeded away from, rather than through, the recombination site. Transcription-stimulated gene targeting was also observed when single-stranded plasmid vectors complementary to either the coding or template strand were used as recombination substrates. Our results indicate that gene targeting, like other forms of DNA recombination, can be stimulated by target site transcription. The implications of our observations on current models of transcription-stimulated recombination are discussed.
Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful ... more Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins which were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI +-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.
V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2... more V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex plays a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Herein we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V (D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly 10-fold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins significantly reduced the fidelity of signal joint recombination in wild-type cells. The latter finding was somewhat unexpected because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells dramatically reduced the fidelity of signal joint recombination. These data reveal a heretofore unreported role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.
The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophospha... more The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan, is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here we show that the DNA repair protein, O 6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32 P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with guanine half mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT-a variant containing a single point mutation in the protein's active site-was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half mustards. HPLC-ESI +-MS/MS sequencing of the tryptic peptides obtained from the wild type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys 145 and Cys 150). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid and N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically.
Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to ... more Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of druginduced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O 6-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Lower numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.
The data in the present study show that DNA polymerase γ and DNA ligase III interact in mitochond... more The data in the present study show that DNA polymerase γ and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase γ was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase γ with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretat...
The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the ... more The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (∆dnl4). The Escherichia coli EcoRI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant EcoRI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and ∆dnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.
1,2,3,4-Diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate car... more 1,2,3,4-Diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate carcinogenic metabolite of the common industrial chemical and environmental carcinogen 1,3butadiene. DEB is a bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs), which are thought to play a central role in its biological activity. Previous studies with recombinant proteins have shown that the biological outcomes of DEB-induced DPCs are strongly influenced by protein identities. The present work combines affinity capture methodology with mass spectrometry-based proteomics and immunological detection to identify the proteins which form DPCs in nuclear extracts from human cervical carcinoma (HeLa) cells. We identified 39 human proteins that form covalent DPCs in the presence of DEB. DNA-protein cross-linking efficiency following treatment with 25 mM DEB was 2-12%, depending on protein identity. HPLC-ESI +-MS/MS analysis of the total proteolytic digests of cross-linked proteins revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3butanediol conjugates, suggesting that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA.
We examined unequal homologous DNA recombination between human repetitive DNA elements located on... more We examined unequal homologous DNA recombination between human repetitive DNA elements located on a yeast artificial chromosome (YAC) and transforming plasmid molecules. A plasmid vector containing an Alu element, as well as a sequence identical to a unique site on a YAC, was introduced into yeast and double recombinant clones analyzed. Recombination occurs between vector and YAC Alu elements sharing as little as 74% identity. The physical proximity of an Alu element to the unique DNA segment appears to play a significant role in determining the frequency with which that element serves as a recombination substrate. In addition, cross-over points of the recombination reaction are largely confined to the ends of the repetitive element. Since a similar distribution of crossover sites occurs during unequal homologous recombination in human germ and somatic tissue, we propose that similar enzymatic processes may be responsible for the events observed in our system and in human cells. This suggests that further examination of the enzymology of unequal homologous recombination of human DNA within yeast may yield a greater understanding of the molecular events which control this process in higher eukaryotes.
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Papers by Colin Campbell