Search

see attached

1,1,2,2- Cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980–995). Interestingly, cisplatin induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI(+)-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu(110), Lys(125), Cys(145), His(146), Arg(147), and Cys(150). Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI(+)-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH(3))(2){d(GpG)}] cross-links which were detected by HPLC-ESI(+)-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.

The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N6-(2-hydroxy-3-buten-1-yl)-2′-deoxyadenosine (N6-HB-dA), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-HMHP-dA), and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (N6,N6-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2′-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (S)-N6-HB-dA, (R,S)-1,N6-HMHP-dA, and (R,R)-N6,N6-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (S)-N...

While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways is elusive. Herein a PCR-based assay was employed to demonstrate that plasmids containing DNA-protein crosslinks are rapidly repaired following electroporation into isolated mammalian mitochondria. Several lines of evidence argue that this repair occurs via homologous recombination. First, DNA-protein crosslinks present on plasmid DNA homologous to the mitochondrial genome were efficiently repaired (21 % repair in three hours), whereas a DNA-protein crosslink present on DNA that lacked homology to the mitochondrial genome remained unrepaired. Second, DNA-protein crosslinks present on plasmid DNA lacking homology to the mitochondrial genome were repaired when they were co-electroporated into mitochondria with an undamaged, homologous plasmid DNA molecule. Third, no repair was observed when DNA-protein crosslink-containing plasmids were electroporated into mitochondria isolated from cells pre-treated with the Rad51 inhibitor B02. These findings suggest that mitochondria utilize homologous recombination to repair endogenous and xenobiotic-induced DNA-protein crosslinks. Consistent with this interpretation, cisplatin-induced mitochondrial DNA-protein crosslinks accumulated to higher levels in cells pre-treated with B02 than in control cisplatin-treated cells. These results represent the first evidence of how spontaneous and xenobiotic-induced DNA-protein crosslinks are removed from mitochondrial DNA.

Due to evolving population demographics, altered exposures, and new technology, new cohorts need to be initiated. Here we report our efforts from a pilot cohort study of the 10,000 Families Study (10KFS). The goal of the pilot is to establish the infrastructure, protocols, and standard operating procedures for a larger study that would provide the data to further our understanding of the determinants of health across the life course and intergenerational transmission of environmental, genetic, and epigenetic risk factors. We have focused on cancer, as this is the leading cause of death in Minnesota. Data collection methods include online questionnaires and attendance at a health fair for various physical and biologic measurements. For this family-based study, a family is eligible if at least one member of two, preferably three, generations enrolls, and each member is willing, or willing for their child(ren), to provide DNA from blood or saliva. To facilitate eventual scale-up of the cohort, we developed and implemented a database system to electronically manage eligibility screening, enrollment, questionnaire administration, and health fair scheduling. Family enrollment is initiated when an index participant is screened for eligibility online. When the database detects a new eligible index participant, custom scripts execute a set of actions that includes sending emails to the index participant with instructions for family expansion, sending links for the online questionnaire to all family members who join, scheduling appointments at upcoming health fairs, and any reminders for steps not completed. Automated reports are generated nightly to update the investigator website about enrollment, data completeness, and follow-up. We have pilot tested our recruitment procedures with individuals attending the 2017 and 2018 Minnesota State Fair, one of the largest public gatherings in the upper Midwest. To date, 1,273 index participants have been screened. Of these, 151 successfully enrolled another eligible family member for a total of 519 participants. Thus far, 138 participants have attended a health fair. Participant ages range from less than a year to over 90 (median 48 among adults and 7 among children). Next, we intend to expand recruitment methods including traditional and social media, develop an electronic remote consent process, establish electronic feeds of medical records from Minnesota healthcare systems, and create linkages with the state’s cancer registry. Citation Format: DeAnn Lazovich, Colin Campbell, Heather Nelson, Jen Poynter, Anna Prizment, Sara Putnam, Cavan Reilly, Michelle Roesler, Logan Spector, Bharat Thyagarajan. Establishing feasibility of a new cohort: The 10,000 Families Study [abstract]. In: Proceedings of the AACR Special Conference on Modernizing Population Sciences in the Digital Age; 2019 Feb 19-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(9 Suppl):Abstract nr A24.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

N,N-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA-DNA and DNA-protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA-protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI(+)-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage.

DNA-protein crosslinks (DPCs) are complex DNA lesions that induce mutagenesis and cell death. DPCs are created by common antitumor drugs, reactive oxygen species, and endogenous aldehydes. Since these agents create other types of DNA damage in addition to DPCs, identification of the mechanisms of DPC repair is challenging. In this study, we created plasmid substrates containing site-specific DPC lesions, as well as plasmids harboring lesions that are selectively repaired by the base excision or nucleotide excision repair (NER) pathways. These substrates were transfected into mammalian cells and a quantitative real-time PCR assay employed to study their repair. This assay revealed that DPC lesions were rapidly repaired in wild-type human and Chinese hamster derived cells, as were plasmids harboring an oxoguanine residue (base excision repair substrate) or cholesterol lesion (NER substrate). Interestingly, the DPC substrate was repaired in human cells nearly three times as efficiently as in Chinese hamster cells (>75% vs ∼25% repair at 8 h post-transfection), while there was no significant species-specific difference in the efficiency with which the cholesterol lesion was repaired (∼60% repair). Experiments revealed that both human and hamster cells deficient in NER due to mutations in the xeroderma pigmentosum A or D genes were five to ten-fold less able to repair the cholesterol and DPC lesions than were wild-type control clones, and that both the global genome and transcription-coupled sub-pathways of NER were capable of repairing DPCs. In addition, analyses using this PCR-based assay revealed that a 4 kDa peptide DNA crosslink was repaired nearly twice as efficiently as was a ∼38 kDa DPC, suggesting that proteolytic degradation of crosslinked proteins occurs during DPC repair. These results highlight the utility of this PCR-based assay to study DNA repair and indicate that the NER machinery rapidly and efficiently repairs plasmid DPC lesions in mammalian cells.

Platinum-based antitumor drugs such as 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin), carboplatin, and oxaliplatin are currently used to treat nearly 50% of all cancer cases, and novel platinum based agents are under development. The antitumor effects of cisplatin and other platinum compounds are attributed to their ability to induce interstrand DNA-DNA cross-links, which are thought to inhibit tumor cell growth by blocking DNA replication and/or preventing transcription. However, platinum agents also induce significant numbers of unusually bulky and helix-distorting DNA-protein cross-links (DPCs), which are poorly characterized because of their unusual complexity. We and others have previously shown that model DPCs block DNA replication and transcription and cause toxicity in human cells, potentially contributing to the biological effects of platinum agents. In the present work, we have undertaken a system-wide investigation of cisplatin-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells using mass spectrometry-based proteomics. DPCs were isolated from cisplatin-treated cells using a modified phenol/chloroform DNA extraction in the presence of protease inhibitors. Proteins were released from DNA strands and identified by mass spectrometry-based proteomics and immunological detection. Over 250 nuclear proteins captured on chromosomal DNA following treatment with cisplatin were identified, including high mobility group (HMG) proteins, histone proteins, and elongation factors. To reveal the exact molecular structures of cisplatin-mediated DPCs, isotope dilution HPLC-ESI+-MS/MS was employed to detect 1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2′-deoxyguanosine-7-yl)-platinum (II) (dG-Pt-Lys) conjugates between the N7 guanine of DNA and the ε-amino group of lysine. Our results demonstrate that therapeutic levels of cisplatin induce a wide range of DPC lesions, which likely contribute to both target and off target effects of this clinically important drug.

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 drug-induced 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. Smaller 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.

1,2,3,4-Diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds.

Background Fanconi anemia (FA) patients are hypersensitive to DNA alkylating agents and require lower doses than non-FA patients to minimize serious toxicity. The mechanism by which hypersensitivity occurs is thought to be due to the inability of these individuals to effectively repair drug-induced interstrand DNA–DNA crosslinks. We recently developed a highly sensitive assay for cyclophosphamide specific interstrand DNA–DNA crosslinks (G-NOR-G) and are able to quantify and compare formation of these adducts in the blood of patients. Therefore we sought to determine whether FA patients have higher in vivo exposure to the cyclophosphamide specific interstrand DNA crosslink, G-NOR-G, relative to patients without FA. Procedure Cyclophosphamide interstrand DNA crosslinks were measured with the first dose of cyclophosphamide in FA and non-FA patients receiving a cyclophosphamide based preparative regimen prior to hematopoietic cell transplantation (HCT). FA patients received a lower cyclophosphamide dose than the non-FA patients (5–10 mg/kg/day vs. 50–60 mg/kg/day). Results Despite the lower cyclophosphamide dose and lower plasma concentrations in FA patients, they had G-NOR-G amounts similar to the non-FA patients (area under the curve (AUC)0–∞, 99.8 vs. 144.9 G-NOR-G adducts/106 nucleotides hour, respectively, P = 0.47). When G-NOR-G AUC was normalized for cyclophosphamide plasma concentrations, FA study subjects produced 15-fold higher adducts than non-FA patients (P = 0.05). Conclusions FA patients are hypersensitive to DNA alkylating agents possibly as a result of greater formation of cyclophosphamide specific interstrand DNA crosslinks and/or diminished capacity for DNA repair. Identification and quantification of these adducts may be important determinant of cyclophosphamide related toxicity. Pediatr Blood Cancer 2012; 58: 708–714. © 2011 Wiley Periodicals, Inc.

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 1,3-butadiene. 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 that 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. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (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)-2,3-butanediol conjugates, suggesting that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA.

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.

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 double-strand 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.

Fibroblasts from patients with Fanconi anemia (FA) display genomic instability, hypersensitivity to DNA cross-linking agents, and deficient DNA end joining. Fibroblasts from two FA patients of unidentified complementation group also had significantly increased cellular homologous recombination (HR) activity. Results described herein show that HR activity levels in patient-derived FA fibroblasts of groups A, C, and G were 10-fold greater than those seen in normal fibroblasts. In contrast, HR activity in group D2 fibroblasts was identical to that in normal cells. Western blot analysis revealed that the RAD51 protein was elevated 10-fold above normal levels in group A, C, and G fibroblasts, but was not altered in group D2 fibroblasts. HR activity levels in these former cells could be restored to near-normal levels by electroporation with anti-RAD51 antibody, whereas similar treatment of normal and complementation group D2 fibroblasts had no effect. These findings are consistent with a model in which FA proteins function to coordinate DNA double-strand break repair activity by regulating both recombinational and non-recombinational DNA repair. Interestingly, whereas positive regulation of DNA end joining requires the combined presence of all FA proteins thus far tested, suppression of HR, which is minimally dependent on the FANCA, FANCC, and FANCG proteins, does not require FANCD2.

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.

Xenobiotic-induced interstrand DNA–DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the XPA or FANCD2 genes. Comet assays and γ-H2AX focus analyses conducted with hamster cells revealed that ICL removal was associated with chromosomal double strand break formation, and that these breaks persisted in V-H4 cells as compared to control cells. Our findings suggest that ICL repair in cells with defects in the Fanconi anemia repair pathway is associated with aberrant re-joining of repair-induced double strand breaks, potentially resulting in lethal chromosome rearrangements.

The Emily Bay archaeological molluscan fauna as an ensemble is almost entirely intertidal in its natural occurrence, with seven species preferring sand or mud substrates and 13 species preferring hard substrates. The only exceptions are the pelagic cephalopods Nautilus and Spirula. The gastropod species Nerita atramentosa is dominant in both numbers and by weight. The rocky intertidal platform was the focus of mollusc collecting. The four most common species derive from this zone and habitually cluster in colonies, which would have made them a preferred prey. Among the many factors that may have contributed to eventual abandonment of Norfolk Island, a scarcity of easily harvestable coastal marine resources would probably have been significant.

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 (Δdnl4). 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 Δdnl4 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.

Mammalian mitochondrial DNA end-binding activity is nearly indistinguishable from that of nuclear Ku. This observation led to the hypothesis that mitochondrial DNA end-binding activity is in part dependent upon Ku80 gene expression. To test this hypothesis, we assayed for Ku activity in mitochondrial extracts prepared from the xrs-5 hamster cell line that lacks Ku80 mRNA expression. Mitochondrial protein extracts prepared from this cell line lacked the DNA end-binding activity found in similar extracts prepared from wild-type cells. Azacytidine-reverted xrs-5 cells that acquired nuclear DNA end-binding activity also acquired mitochondrial DNA end-binding activity. Western blot analysis of human mitochondrial protein extracts using a monoclonal antibody specific for an N-terminal epitope of Ku80 identified a protein with an apparent molecular weight of 68 kDa. This mitochondrial protein was not detected by a monoclonal antibody specific for an epitope at the C-terminal end of Ku80. Consistently, while both the N- and C-terminal Ku80 monoclonal antibodies supershifted the nuclear DNA end-binding complex on an electrophoretic mobility shift assay, only the N-terminal monoclonal antibody supershifted the mitochondrial DNA end-binding complex. To confirm that the 68 kDa Ku protein was not a consequence of nuclear protein contamination of mitochondrial preparations, highly purified intact nuclei and mitochondria were treated with proteinase K which traverses the pores of intact nuclei but gains limited access into intact mitochondria. Ku80 in purified intact nuclei was sensitive to treatment with this protease, while the 68 kDa Ku protein characteristic of purified intact mitochondria was resistant. Further, immunocytochemical analysis revealed the co-localization of the N-terminal specific Ku80 monoclonal antibody with a mitochondrial-targeted green fluorescence protein. Mitochondrial localization of the C-terminal Ku80 monoclonal antibody was not observed. These data are consistent with the hypothesis that a C-terminally truncated form of Ku80 is localized in mammalian mitochondria where it functions in a DNA end-binding activity.

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 discuss the potential usefulness of the ciliates' characteristic nuclear duality for further analyses of the potentially unique roles of Tetrahymena RAD51.

Gene transfer into neuronal cells provides an important approach to study their function. Particle-mediated gene delivery was used to transfect rat dorsal root ganglion (DRG) and hippocampal neurons in primary culture with the genes for the enhanced blue and green fluorescent proteins (EBFP and EGFP) under control of the cytomegalovirus promoter. Quantitative analysis of marker protein fluorescence detected expression at 3 h that continued to increase for 48 h. For DRG neurons the optimal expression efficiency of 8+/-2% was obtained 24 h following transfection. In contrast, approximately 2+/-1% of hippocampal neurons in culture expressed EGFP at 3 h which subsequently declined. Co-transfection of DRG cultures with two plasmids produced reliable expression of both genes. Transfected DRG neurons exhibited normal electrophysiological properties, and resting and stimulated intracellular Ca2+ concentrations were unchanged. After transfection, 44% of hippocampal neurons remained in functional synaptic networks as indicated by glutamatergic Ca2+ spiking activity. Particle-mediated gene delivery provided a straightforward, reproducible and efficient method for transfection of neurons in primary culture. Transfected cells were easily identified by EGFP fluorescence, enabling subsequent physiological analysis. Biolistic particle bombardment was well tolerated by peripheral neurons, although caution was required when this method was applied to CNS cultures.

We purified to near homogeneity a previously identified 100 kDa mammalian homologous DNA pairing protein. The purified 100 kDa protein also catalyzed high levels of cell-free homologous DNA recombination activity. This ATP-dependent activity was capable of forming conservative recombinant products between two circular, double-stranded DNA molecules. We were unable to detect any DNA polymerase, DNA ligase, or 5' or 3' exonuclease activity associated with this purified material. The purified 100 kDa protein bound silver nitrate as well as a monoclonal antibody specific for nucleolin. A recombinant protein comprised of the Escherichia coli maltos-ebinding protein fused to the carboxyl-terminal two-thirds of human nucleolin possessed homologous DNA pairing activity. These data indicate that the 100 kDa homologous DNA pairing protein is nucleolin. The observation that nucleolin can carry out homologous DNA strand pairing in vitro raises the prospect that it may function similarly in vivo.

The RAD51 gene is a eukaryotic homolog of rec A, 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/m2. 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.

Physiological and pathological Ca2+loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+into mitochondria. Mobilization of Ca2+stores with bradykinin (BK) increased the cytosolic Ca2+concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2+-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+concentration ([Ca2+]mt) of 2.0 ± 0.2 μM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mtincrease, although it did not affect the [Ca2+]ctransient. Basal [Ca2+]cand [Ca2+]mtin control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 ± 1% in normal cells to 19 ± 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mttransients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.

1,3-Butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a capillary HPLC-high resolution ESI⁺-MS/MS (HPLC-ESI⁺-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafiltration, solid-phase extraction, and isotope dilution HPLC-ESI⁺-HRMS/MS analysis on an Orbitrap Velos mass spectrometer. Following method validation, N7-THBG was quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of DEB and in DNA isolated from blood of smokers, nonsmokers, individuals participating in a smoking cessation program, and occupationally exposed workers. N7-THBG concentrations increased linearly from 31.4 ± 4.84 to 966.55 ± 128.05 adducts per 10⁹ nucleotides in HT1080 cells treated with 1-100 μM DEB. N7-THBG amounts in leukocyte DNA of nonsmokers, smokers, and occupationally exposed workers were 7.08 ± 5.29, 8.20 ± 5.12, and 9.72 ± 3.80 adducts per 10⁹ nucleotides, respectively, suggesting the presence of an endogenous or environmental source for this adduct. The availability of sensitive HPLC-ESI⁺-HRMS/MS methodology for BD-induced DNA adducts in humans will enable future population studies of interindividual and ethnic differences in BD bioactivation to DNA-reactive epoxides.

We have characterized homologous DNA recombination activity in nuclear protein extracts prepared from quiescent and regenerating rat livers. Activity measured in regenerating liver extracts was elevated approximately 35-fold above control, and its appearance closely mirrored the first wave of DNA synthesis, peaking 24 hours after a regenerative stimulus, and returning fairly rapidly to basal levels. We also identified a strand-transferase protein of approximately 100 kDa whose presence in these extracts correlates with homologous recombination activity. Recent evidence suggests that mammalian somatic cells possess a recombinational DNA repair mechanism analogous to that described in the yeast Saccharomyces cerevisiae. Our results indicate that this recombinational repair process may be regulated in vivo by, or play a role in, progression through the cell division cycle.

A human testis cDNA library was screened with a hybridization probe encoding the mouse RAD52 gene. Two classes of clones were identified, one derived from the human RAD52 homolog (hRAD52), the other derived from a pseudogene. In addition to many point mutations, several of which encode stop codons, the pseudogene contains a number of frame shifts and a 103-bp deletion. We further determined that the pseudogene is processed and is located on human chromosome 2, in contrast to hRAD52 which is found on chromosome 12. Reverse transcription-PCR analysis of cultured human diploid fibroblasts, as well as fibrosarcoma cells, revealed that while hRAD52 is expressed at low, but detectable levels in these cells, the pseudogene is not

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.

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 that 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-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.

Nitrogen mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA-DNA cross-links. However, nitrogen mustards are able to produce a variety of other lesions, including DNA-protein cross-links (DPCs). DPCs induced by nitrogen mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple nitrogen mustard, mechlorethamine. Following incubation of 5'-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chromatography-electrospray tandem mass spectrometry of tryptic peptides. Mechlorethamine treatment resulted in the formation of DPCs with nuclear proteins involved in chromatin regulation, DNA replication and repair, cell cycle control, transcriptional regulation, and cell architecture. Western blot analysis was employed to confirm protein identification and to quantify the extent of drug-mediated cross-linking. Mass spectrometry of amino acid-nucleobase conjugates found in total proteolytic digests revealed that mechlorethamine-induced DPCs are formed via alkylation of the N7 position of guanine in duplex DNA and cysteine thiols within the proteins to give N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine lesions. The results described herein suggest that cellular exposure to nitrogen mustards leads to cross-linking of a large spectrum of nuclear proteins to chromosomal DNA, potentially contributing to the cytotoxic and mutagenic effects of these drugs.

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.

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 interpretation that the interaction of DNA ligase III and DNA polymerase γ is required for proper maintenance of the mammalian mitochondrial genome.

Fanconi anemia (FA) is an autosomal recessive disorder associated with pancytopenia and cancer susceptibility. The disorder is heterogeneous, with at least nine complementation groups having been identified. Several recent studies have suggested that defective plasmid DNA end-joining is a consistent feature of FA cells. It was therefore surprising to discover a strain of fibroblasts from an FA patient that possessed wild-type plasmid DNA end-joining activity. Unlike other FA strains, these fibroblasts have wild-type levels of homologous DNA recombination activity and are relatively insensitive to restriction endonuclease-induced death. Interestingly, while end-joining in a number of FA fibroblast strains belonging to complementation groups A, C, and D2 was approximately 70% precise, end-joining in this latter strain of fibroblasts was more than 95% imprecise. Analysis revealed that these latter cells harbored an allele of the FA C gene, referred to as 322delG, that encodes an amino-terminal truncated protein. The relative rarity of this allele precluded the analysis of other FA fibroblast strains; however, studies revealed that overexpression of this allele in normal cells recapitulated the DNA end-joining phenotype seen in the 322delG FA fibroblast strain. These results indicate that DNA end-joining in fibroblasts expressing the 322delG allele of the FA-C gene in fibroblasts is highly imprecise; however, the DNA repair efficiency of these cells is more normal than that commonly associated with FA fibroblasts. This conclusion is intriguing, since a number of reports have suggested that patients harboring this allele exhibit a milder clinical course than do individuals with other alleles of the FA-C gene.

Activation of NMDA receptors produces large increases in cytosolic Ca(2+) that are taken up into mitochondria. We used recombinant aequorin targeted to mitochondria to report changes in matrix Ca(2+) in rat hippocampal neurons in culture. Upon binding Ca(2+), aequorin emits a photon in a one-shot reaction that consumes the indicator. Here we show that stimulation with NMDA produced a mitochondrial Ca(2+) response that rapidly inactivated. However, following a 30-min recovery period the response was restored, suggesting the presence of a pool of indicator that was not exposed to high Ca(2+) during the initial stimulus. We speculate that aequorin distant from the Ca(2+) source was protected from microdomains of high Ca(2+) near the plasmalemma and that this aequorin moved, either by movement of individual mitochondria or via the mitochondrial tubular network, to replenish consumed indicator during the recovery time. A large Ca(2+) increase in a subset of mitochondria could produce local changes in energy metabolism, regional Ca(2+) buffering, and foci that initiate neurotoxic processes.

Flavopiridol (FP) is an investigational new drug currently in Phase II clinical trials for the treatment of solid tumors (Senderowicz et al., 1998). The precise mechanism of action of this compound is unknown, however, it is known to inhibit several members of the cyclin-dependent protein kinase family, and to induce cell death (Senderowicz, 1999, Schrump, et al., 1998, Bible and Kaufmann, 1996). The objective of this proposal is to gain insight into the molecular mechanism(s) whereby human tumor cells become resistant to FP. To gain greater insight into this question, a FP-resistant clone was generated from the human MCF-7 breast adenocarcinoma cell line. This clone was obtained by exposing growing cultures of MCF-7 cells to increasing concentrations of FP over a several month period of time. The resulting clone, named MCF-71F, has an IC50 for FP that is 24-fold lower than that of the parental MCF-7 cell line cell line from which it was derived. Specifically, the experiments described in this proposal are designed to identify the molecular basis for FP resistance in MCF-7/FP cells. We have evaluated, or are in the process of evaluating cellular levels of known FP targets, i.e. cyclin-dependent protein kinases in MCF-7/FP and MCF-7 cells. In addition, we have begun to examine the relative resistance of these cells to a number of drugs. Finally, we have been examining the relative expression levels of a number of drug efflux pumps in these cell lines. It is anticipated that FP or FP-like molecules will ultimately assume a place in the modern cancer chemotherapeutic armamentarium. Thus, insight gained into the molecular basis of FP drug resistance in MCF-7/FP cells may ultimately prove beneficial in the design of second or third generation FP analogues. It is also conceivable that this information may aid in the development of chemotherapy strategies to minimize the emergence of clinical resistance to these agents.

Next Section Abstract A yeast two-hybrid screen identified the regulatory subunit of the calcium-dependent protease calpain as a putative DNA ligase III-binding protein. Calpain binds to the N-terminal region of DNA ligase III, which contains an acidic proline, aspartate, serine, and threonine (PEST) domain frequently present in proteins cleaved by calpain. Recombinant DNA ligase III was a substrate for calpain degradation in vitro. This calpain-mediated proteolysis was calcium-dependent and was blocked by the specific calpain inhibitor calpeptin. Western blot analysis revealed that DNA ligase III was degraded in human fibrosarcoma HT1080 cells following exposure to γ-radiation. The degradation of DNA ligase III was prevented by pretreatment with calpeptin, which protected irradiated cells from death. Calpeptin treatment also blocked 9-amino camptothecin-induced DNA ligase III proteolysis and simultaneously protected the cells from death. HT1080 clones expressing a modified DNA ligase III that lacked a recognizable PEST domain were significantly more resistant to killing by γ-radiation or 9- amino camptothecin than were cells that overexpressed the wild-type form of DNA ligase III. These data show that calpain-mediated proteolysis of DNA ligase III plays an essential role in DNA damage-induced cell death in human cells.

Expression vectors were created in which the 5′ end of the Saccharomyces cerevisiae CDC9 gene, which encodes a mitochondrial targeting peptide, was cloned in-frame with the coding regions of the Eco R I, Hind III, and Pst I endonuclease genes. Expression of the Eco R I and Hind III fusion proteins inhibited growth of yeast on glycerol-containing media and resulted in the nearly quantitative restriction digestion of their mitochondrial DNA. In contrast, expression of Pst I, which does not recognize any sites within yeast mitochondrial DNA, had no effect on growth in glycerol-containing media, and did not affect the integrity of the mitochondrial genome.

Fanconi anemia (FA) is a genetic disorder associated with genomic instability and cancer predisposition. Cultured cells from FA patients display a high level of spontaneous chromosome breaks and an increased frequency of intragenic deletions, suggesting that FA cells may have deficiencies in properly processing DNA double strand breaks. In this study, an in vitro plasmid DNA end joining assay was used to characterize the end joining capabilities of nuclear extracts from diploid FA fibroblasts from complementation groups A, C, and D. The Fanconi anemia extracts had 3–9-fold less DNA end joining activity and rejoined substrates with significantly less fidelity than normal extracts. Wild-type end joining activity could be reconstituted by mixing FA-D extracts with FA-A or FA-C extracts, while mixing FA-A and FA-C extracts had no effect on end joining activity. Protein expression levels of the DNA-dependent protein kinase (DNA-PK)/Ku-dependent nonhomologous DNA end-joining proteins Xrcc4, DNA ligase IV, Ku70, and Ku86 in FA and normal extracts were indistinguishable, as were DNA-dependent protein kinase and DNA end binding activities. The end joining activity as measured by the assay was not sensitive to the DNA-PK inhibitor wortmannin or dependent on the nonhomologous DNA end-joining factor Xrcc4. However, when DNA/protein ratios were lowered, the end joining activity became wortmannin-sensitive and no difference in end joining activity was observed between normal and FA extracts. Taken together, these results suggest that the FA fibroblast extracts have a deficiency in a DNA end joining process that is distinct from the DNA-PK/Ku-dependent nonhomologous DNA end joining pathway.

Hamster EM9 cells, which lack Xrcc1 protein, have reduced levels of DNA ligase III and are defective in nuclear base excision repair. The Xrcc1 protein stabilizes DNA ligase III and may even play a direct role in catalyzing base excision repair. Since DNA ligase III is also thought to function in mitochondrial base excision repair, it seemed likely that mitochondrial DNA ligase III function would also be dependent upon Xrcc1. However, several lines of evidence indicate that this is not the case. First, western blot analysis failed to detect Xrcc1 protein in mitochondrial extracts. Second, DNA ligase III levels present in mitochondrial protein extracts from EM9 cells were indistinguishable from those seen in similar extracts from wild-type (AA8) cells. Third, the mitochondrial DNA content of both cell lines was identical. Fourth, EM9 cells displayed no defect in their ability to repair spontaneous mitochondrial DNA damage. Fifth, while EM9 cells were far more sensitive to the cytotoxic effects of ionizing radiation due to a defect in nuclear DNA repair, there was no apparent difference in the ability of EM9 and AA8 cells to restore their mitochondrial DNA to pre-irradiation levels. Thus, mitochondrial DNA ligase III function is independent of the Xrcc1 protein.

Mammalian mitochondrial protein extracts possess DNA end-binding (DEB) activity. Protein binding to a 394 bp double-stranded DNA molecule was measured using an electrophoretic mobility shift assay. Mitochondrial DEB activity was highly specific for linear DNA. Inclusion of a vast excess of non-radioactive circular DNA did not disrupt binding to radioactive f394. In contrast, binding was abolished by the inclusion of linear competitor DNA. In mammals, nuclear DEB activity is due to Ku, a hetero-dimer composed of the Ku70 and Ku86 proteins. To determine whether mitochondrial DEB activity was also due to Ku, protein extracts were prepared from the Chinese hamster XR-V15B cell line, which lacks this protein. As anticipated, nuclear extracts prepared from these cells lacked DEB activity. In contrast, mitochondrial extracts prepared from these cells had wild-type levels of DEB activity, demonstrating that this latter activity is not a consequence of nuclear contamination. Although the nuclear and mitochondrial DEB activities are independent of each other, they are nevertheless closely related, since mitochondrial DEB activity was 'supershifted' by both anti-Ku70 and anti-Ku86 antisera. The nuclear DEB protein Ku plays an essential role in nuclear DNA double-strand break repair. The DEB activity described herein may therefore play a similar role in mitochondrial DNA repair.

DNA end-joining was measured by incubating linearized plasmid DNA with mitochondrial protein extracts. A spectrum of end-joined molecules ranging from re-circularized monomer to dimer and higher molecular weight forms was observed. The DNA end-joining reaction required ATP and Mg2+, and was inhibited by sodium chloride. Both cohesive- and blunt-ended DNA molecules were end-joined, although the former were more efficient substrates. Molecular analysis of rejoined molecules revealed that >95% of the linearized DNA were precisely end-joined. The few imprecisely end-joined molecules recovered, sustained deletions that spanned direct repeat sequences. The deletions observed are strikingly similar to those present in mitochondrial genomes of patients with Kearns-Sayre or Pearson syndromes, certain ophthalmic myopathies and the aged. These results suggest that mammalian mitochondria possess a DNA double strand break repair activity similar to that seen in the nucleus, and that this repair pathway may play a role in the generation of mitochondrial DNA deletions associated with a number of human pathologies.

It is widely believed that Fanconi anemia cells possess a reduced ability to repair inter-strand DNA cross-links. While the mechanism through which inter-strand DNA cross-links are removed from mammalian chromosomes is unknown, these lesions are repaired via homologous recombination in lower eukaryotes and bacteria. Based on the hypothesis that a similar mechanism of DNA repair functions in mammalian somatic cells, we measured homologous recombination activity in diploid fibroblasts from healthy donors, and Fanconi anemia patients. Somewhat surprisingly, homologous recombination levels in nuclear protein extracts prepared from Fanconi anemia cells were nearly 100-fold higher than in extracts prepared from control cells. We observed a similar increase in the activity of a 100-kDa homologous DNA pairing protein in extracts from Fanconi anemia cells. Transfection studies confirmed that plasmid homologous recombination levels in intact Fanconi anemia cells were substantially elevated, compared with control cells. These results suggest that inappropriately elevated levels of homologous recombination activity may contribute to the genomic instability and cancer predisposition that characterize Fanconi anemia.

Mitochondrial protein extracts from normal and immortalized mammalian somatic cells catalyze homologous recombination of plasmid DNA substrates. Mitochondrial homologous recombination activity required exogenous adenosine triphosphate, although substantial activity remained when non-hydrolyzable analogs were used instead. There was no requirement for added nucleoside triphosphates, and the reaction was not inhibited by dideoxyadenosine triphosphate or aphidicolin. The majority of recombinant plasmid molecules result from a conservative process, indicating that nuclease-mediated strand-annealing is not responsible for the mitochondrial homologous recombination activity. Affinity-purified anti-recA antibodies inhibited the reaction, suggesting that activity is dependent on a mammalian mitochondrial homolog of the bacterial strand-transferase protein. The presence of homologous recombination activity within mammalian mitochondrial extracts suggests that this process is involved in mitochondrial DNA repair.

The Saccharomyces cerevisiae RAD52 gene was introduced into the human fibrosarcoma-derived cell line HT1080. Transfected cell lines that expressed the yeast transgene catalyzed inter-plasmid homologous DNA recombination at frequencies approx. 12-fold higher than did control cells. Additional experiments revealed that yeast RAD52 gene expression increased the level of resistance to the DNA damaging agents diepoxybutane, and methyl methanesulfonate, but did not alter sensitivity to ultraviolet radiation. These results indicate that the S. cerevisiae Rad52 protein can function in a human somatic cell background and provide support for the idea that a homologous recombination-based DNA repair process functions in mammalian somatic cells.

We evaluate the effect of target site transcription on gene targeting in cultured human fibrosarcoma 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.

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs the mutagenic O6-methylguanine (O6-mG) lesion by transferring a methyl group from the 6-position of guanine to a cysteine residue on the protein. The simplest possible mechanism is an SN2 process in which the cysteine displaces the methyl group off of the guanine in a concerted reaction. To probe the interactions between the protein and guanine leaving group, oligodeoxynucleotide duplexes containing analogs of O6mG were synthesized and then reacted with AGT. The analogs, which were incorporated into deoxynucleotides include O6-methylhypoxanthine (O6-mH),S6-methyl-6-thioguanine (S6mG),S6-methyl-6-thiohypoxanthine (S6mH),Se6-methyl-6-selenoguanine (Se6mG),Se6-methyl-6-selenohypoxanthine (Se6mH), O6-methyl-1-deazaguanine (O6m1DG), O6-methyl-3-deazaguanine (O6m3DG), and O6-methyl-7-deazaguanine (O6m7DG), differ from O6mG in that the heteroatoms have been replaced so that they are poorer hydrogen bond participants and proton acceptors. AGT was reacted with oligonucleotide duplexes of the sequence 5'-GGC GCT XGA GGC GTG-3' in which X was O6mG or an analog in which X was paired with C. The reactions in 50 mM Tris-HCl and 1 mM EDTA, pH7.6 and 37 degrees C, were followed by anion-exchange HPLC in 10 mM NaOH with a NaCl gradient. All detected reactions were demethylations of the oligodeoxynucleotides except for O6m3DG, which reacted in an unknown manner.(ABSTRACT TRUNCATED AT 250 WORDS)