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1.
Leukemia ; 32(1): 49-60, 2018 01.
Article in English | MEDLINE | ID: mdl-28579617

ABSTRACT

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11-Rad51-DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair 'addiction' and provide an effective therapeutic option for Ph+ B-cell precursor ALL.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , DNA Repair/drug effects , Fusion Proteins, bcr-abl/metabolism , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 2/antagonists & inhibitors , Philadelphia Chromosome/drug effects , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Animals , Cell Line, Tumor , DNA Breaks, Double-Stranded/drug effects , Doxorubicin/administration & dosage , Humans , Mice , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism
2.
Pregnancy Hypertens ; 4(3): 235-6, 2014 Jul.
Article in English | MEDLINE | ID: mdl-26104623

ABSTRACT

OBJECTIVE: 1. To examine relationships between 25-hydroxy vitamin D (25OHD) in women with type 1 diabetes (T1DM) during pregnancy, post-delivery and in cord blood. 2. To investigate interactions between maternal body mass index (BMI) and foetal vitamin D status. 3. To examine relationships between maternal 25OHD and glycosylated haemoglobin (HbA1c). METHODOLOGY: An observational study of 52 pregnant controls without diabetes and 65 pregnant women with T1DM in a university teaching hospital. 25OHD was measured by liquid chromatography tandem mass spectrometry. RESULTS: Vitamin D deficiency (25OHD <25nmol/L) was apparent in control and T1DM women in all 3 trimesters. All cord blood 25OHD were <50nmol/L. Maternal 25OHD correlated positively with cord 25OHD at all 3 trimesters in the T1DM group (p=0.02; p<0.001; p<0.001). Cord 25OHD was significantly lower for T1D women classified as obese vs. normal weight at booking [normal weight BMI <25kg/m(2) vs. obese BMI〉30kg/m(2) (nmol/L±SD); 19.93±11.15 vs. 13.73±4.74, p=0.026]. In the T1DM group, HbA1c at booking was significantly negatively correlated with maternal 25OHD at all 3 trimesters (p=0.004; p=0.001; p=0.05). CONCLUSION: In T1DM pregnancy, low vitamin D levels persist throughout gestation and post-delivery. Cord blood vitamin D levels correlate with those of the mother, and are significantly lower in obese vs normal weight women. Maternal vitamin D levels exhibit a significant negative relationship with HbA1c, supporting a potential role for this vitamin in maintaining glycaemic control.

3.
Pregnancy Hypertens ; 4(3): 236, 2014 Jul.
Article in English | MEDLINE | ID: mdl-26104627

ABSTRACT

OBJECTIVE: The physiological importance of the C3 epimers of vitamin D (3-epi-25OHD2/3) is uncertain and there have been limited studies determining the levels of these epimers in human populations. The aims of the current study were (1) to determine 3-epi-25OHD2/3 levels throughout non-diabetic and T1DM pregnancy, (2) to examine the relationships between 25OHD and 3-epi-25OHD, (3) to assess the impact of maternal BMI on 3-epi-25OHD and examine associations with markers of glycaemic control. METHODOLOGY: An observational study of 52 pregnant controls without diabetes and 65 pregnant women with T1DM in a university teaching hospital. 25OHD and 3-epi-25OHD were measured by liquid chromatography tandem mass spectrometry. RESULTS: 3-Epi-25OHD was found in 90.2% of control (median 0.9nmol/L; range 0.1-5.9nmol/L), and in 94.5% of T1DM, women (median 1.4nmol/L; range 0.1-10.5nmol/L). In both control and T1DM groups, maternal and cord 3-epi-25OHD correlated significantly with 25OHD. Seasonal variation in maternal 3-epi-25OHD levels was evident in both groups; Summer levels were significantly higher than all other seasons in the control group (p<0.001) and significantly higher than Spring (p=0.003) and Winter (p<0.001) in the T1DM group. In T1DM women HbA1c was significantly negatively correlated with 3-epi-25OHD at trimesters 1 and 2 (p=0.049; p=0.001) and with cord 3-epi-25OHD (p=0.012). Maternal BMI >30kg/m(2) had a significant negative impact on 3-epi-25OHD. CONCLUSION: Maternal 3-epi-25OHD exhibits seasonal variation and, in common with cord 3-epi-25OHD, correlates with 25OHD throughout both non-diabetic and T1D pregnancy. In T1DM women 3-epi-25OHD is associated with a key marker of glycaemic control.

4.
Ultramicroscopy ; 111(3): 207-11, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21333858

ABSTRACT

The electronic characteristics of semiconductor-based devices are greatly affected by the local dopant atom distribution. In Mg-doped GaN, the clustering of dopants at structural defects has been widely reported, and can significantly affect p-type conductivity. We have studied a Mg-doped AlGaN/GaN superlattice using transmission electron microscopy (TEM) and atom probe tomography (APT). Pyramidal inversion domains were observed in the TEM and the compositional variations of the dopant atoms associated with those defects have been studied using APT. Rarely has APT been used to assess the compositional variations present due to structural defects in semiconductors. Here, TEM and APT are used in a complementary fashion, and the strengths and weaknesses of the two techniques are compared.

5.
Rev Sci Instrum ; 81(6): 063701, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20590240

ABSTRACT

We have employed an atomic force microscope with a high sampling rate to image GaN samples grown using an epitaxial layer overgrowth technique and treated with silane and ammonia to enlarge the surface pits associated with threading dislocations (TDs). This allows TDs to be identified in high pixel density images tens of microns in size providing detailed information about the spatial distribution of the TDs. An automated software tool has been developed, which identifies the coordinates of the TDs in the image. Additionally, we have imaged the same sample using Kelvin probe force microscopy, again at high pixel density, providing data about the local changes in surface potential associated with hundreds of dislocations.

6.
Nucleic Acids Res ; 29(20): 4134-43, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11600702

ABSTRACT

Small looped mispairs are efficiently corrected by mismatch repair. The situation with larger loops is less clear. Repair activity on large loops has been reported as anywhere from very low to quite efficient. There is also uncertainty about how many loop repair activities exist and whether any are conserved. To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays. Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates. Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast. Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology. Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products. These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals.


Subject(s)
DNA Repair , DNA, Fungal/genetics , Saccharomyces cerevisiae/genetics , Base Pair Mismatch , Base Sequence , Genes, Fungal , Mutation , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/metabolism
7.
J Biol Chem ; 276(45): 42588-600, 2001 Nov 09.
Article in English | MEDLINE | ID: mdl-11551933

ABSTRACT

Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase beta (+/+) and (-/-) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U.A, U.G, and U.T targets contained within the lacZalpha gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase beta (+/+) and (-/-) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced ( approximately 88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting approximately 20% of the repair events. Addition of p21 peptide or protein to DNA polymerase beta (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by approximately 2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U.T) DNA was determined to be 5.7-7.2 x 10(-4) when using DNA polymerase beta (+/+) and (-/-) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U.G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U.G target and frequently involved a 1-base deletion or incorporation of dTMP.


Subject(s)
DNA Polymerase beta/physiology , DNA Repair , Mutation , Uracil/metabolism , Animals , Base Sequence , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Mice , Molecular Sequence Data , Proliferating Cell Nuclear Antigen/physiology
8.
Article in English | MEDLINE | ID: mdl-11554295

ABSTRACT

The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 lacZ alpha DNA-based reversion assay. Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZ alpha gene. Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E. coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred. Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides. The misincorporation frequency of BER DNA synthesis at the target site was 5.2 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to G > T to A >> T to C. Uracil-initiated BER DNA synthesis in extracts of E. coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined. Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 x 10(-4). A reduced, but detectable level of BER was observed in extracts of E. coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold.


Subject(s)
Adenocarcinoma/genetics , Colonic Neoplasms/genetics , DNA Glycosylases , DNA Repair/physiology , DNA, Bacterial/genetics , DNA, Neoplasm/genetics , Escherichia coli/genetics , N-Glycosyl Hydrolases/physiology , Neoplasm Proteins/physiology , Uracil/physiology , Viral Proteins/physiology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Aphidicolin/pharmacology , Bacteriophage M13/genetics , Cell Extracts , Cell-Free System , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , DNA Damage , DNA Repair/drug effects , DNA Replication , DNA, Bacterial/metabolism , DNA, Neoplasm/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Escherichia coli/metabolism , Lac Operon/drug effects , Mutation , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Uracil-DNA Glycosidase
9.
J Neuroimaging ; 11(2): 105-11, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11296578

ABSTRACT

OBJECTIVE: Identification of significant asymptomatic carotid artery stenosis (ACAS) is important because of the stroke-risk reduction observed with carotid endarterectomy. The authors developed and validated a simple scoring system based on routinely available information to identify persons at high risk for ACAS using data collected during a community health screening program at various sites in western New York. A total of 1331 unselected volunteers without previous stroke, transient ischemic attack, or carotid artery surgery were evaluated by personal interview and duplex ultrasound. The main outcome measure was carotid artery stenosis > 60% by duplex ultrasound. In the derivation set (n = 887), 4 variables were significantly associated with ACAS > 60%: age > 65 years (odds ratio [OR] = 4.1, 95% confidence interval [CI] = 2.6-6.7), current smoking (OR = 2.0, 95% CI = 1.2-3.5), coronary artery disease (OR = 2.4, 95% CI = 1.5-3.9), and hypercholesterolemia (OR = 1.9, 95% CI = 1.2-2.9). Three risk groups (low, intermediate, and high) were defined on the basis of total risk score assigned on the basis of the strength of association. The scheme effectively stratified the validation set (n = 444); the likelihood ratio and posttest probability for ACAS in the high-risk group were 3.0 and 35%, respectively, and in the intermediate and low-risk groups were 1.4 and 20% and 0.4 and 7%, respectively. Routinely available information can be used to identify persons in the community at high risk for ACAS. Doppler ultrasound screening in this subgroup may prove to be cost-effective and have an effect on stroke-free survival.


Subject(s)
Carotid Stenosis/diagnostic imaging , Mass Screening , Ultrasonography, Doppler, Duplex , Aged , Aged, 80 and over , Carotid Stenosis/epidemiology , Carotid Stenosis/etiology , Comorbidity , Coronary Disease/epidemiology , Coronary Disease/etiology , Cross-Sectional Studies , Female , Humans , Incidence , Male , Middle Aged , Risk Factors
10.
Mol Cell Biol ; 21(3): 940-51, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11154280

ABSTRACT

Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177-3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)(+) mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1 overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1 overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3'-->5' exonuclease activity of replicative DNA polymerases delta and epsilon but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, or PMS1. This suggests that overexpression of MLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K(d) of 3.14 microM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressing MLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.


Subject(s)
Base Pair Mismatch , DNA Repair , Fungal Proteins/genetics , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA Primers/genetics , Dimerization , Fungal Proteins/chemistry , Gene Expression , Gene Silencing , Genes, Fungal , Genome, Fungal , Molecular Sequence Data , MutL Protein Homolog 1 , MutL Proteins , Mutation , Oligonucleotide Array Sequence Analysis , Phenotype , Protein Structure, Quaternary , Sequence Homology, Amino Acid , Suppression, Genetic
11.
J Biol Chem ; 276(3): 2276-85, 2001 Jan 19.
Article in English | MEDLINE | ID: mdl-11035036

ABSTRACT

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung(+) dug(+)), approximately 80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung(-) dug(+)), BH157 (ung(+) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be approximately 5.5 x 10(-)(4) with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.


Subject(s)
DNA Repair , Escherichia coli/genetics , Uracil/metabolism , Base Sequence , Cell Extracts , DNA Primers , DNA Replication
12.
Nucleic Acids Res ; 28(2): 648-54, 2000 Jan 15.
Article in English | MEDLINE | ID: mdl-10606667

ABSTRACT

Mutations in the chromosome 8p WRN gene cause Werner syndrome (WRN), a human autosomal recessive disease that mimics premature aging and is associated with genetic instability and an increased risk of cancer. All of the WRN mutations identified in WRN patients are predicted to truncate the WRN protein with loss of a C-terminal nuclear localization signal. However, many of these truncated proteins would retain WRN helicase and/or nuclease functional domains. We have used a combination of immune blot and immune precipitation assays to quantify WRN protein and its associated 3'-->5' helicase activity in genetically characterized WRN patient cell lines. None of the cell lines from patients harboring four different WRN mutations contained detectable WRN protein or immune-precipitable WRN helicase activity. Cell lines from WRN heterozygous individuals contained reduced amounts of both WRN protein and helicase activity. Quantitative immune blot analyses indicate that both lymphoblastoid cell lines and fibroblasts contain approximately 6 x 10(4)WRN molecules/cell. Our results indicate that most WRN mutations result in functionally equivalent null alleles, that WRN heterozygote effects may result from haploinsufficiency and that successful modeling of WRN pathogenesis in the mouse or in other model systems will require the use of WRN mutations that eliminate WRN protein expression.


Subject(s)
DNA Helicases/metabolism , Werner Syndrome/enzymology , Animals , Blotting, Western , Cell Line, Transformed , Exodeoxyribonucleases , Heterozygote , Humans , Mice , Plasmids , Precipitin Tests , RecQ Helicases , Transfection , Werner Syndrome/genetics , Werner Syndrome/pathology , Werner Syndrome Helicase
14.
J Biol Chem ; 274(25): 17605-11, 1999 Jun 18.
Article in English | MEDLINE | ID: mdl-10364197

ABSTRACT

Single base mispairs and small loops are corrected by DNA mismatch repair, but little is known about the correction of large loops. In this paper, large loop repair was examined in nuclear extracts of yeast. Biochemical assays showed that repair activity occurred on loops of 16, 27, and 216 bases, whereas a G-T mispair and an 8-base loop were poorly corrected under these conditions. Two modes of loop repair were revealed by comparison of heteroduplexes that contained a site-specific nick or were covalently closed. A nick-stimulated repair mode directs correction to the discontinuous strand, regardless of which strand contains the loop. An alternative mode is nick-independent and preferentially removes the loop. Both outcomes of repair were largely eliminated when DNA replication was inhibited, suggesting a requirement for repair synthesis. Excision tracts of 100-200 nucleotides, spanning the position of the loop, were observed on each strand under conditions of limited DNA repair synthesis. Both repair modes were independent of the mismatch correction genes MSH2, MSH3, MLH1, and PMS1, as judged by activity in mutant extracts. Together the loop specificity and mutant results furnish evidence for a large loop repair pathway in yeast that is distinct from mismatch repair.


Subject(s)
Base Pairing/genetics , DNA Repair , Saccharomyces cerevisiae/genetics , Base Pair Mismatch/genetics , DNA Replication/genetics , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes
15.
Biochemistry ; 38(15): 4834-45, 1999 Apr 13.
Article in English | MEDLINE | ID: mdl-10200172

ABSTRACT

The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated. Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung. The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues. Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0. Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer. However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate. Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil. Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP). Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D [Putnam, C. D., Shroyer, M. J. N., Lundquist, A. J., Mol, C. D., Arvai, A. S., Mosbaugh, D. W., and Tainer, J. A. (1999) J. Mol. Biol. 287, 331-346] provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage. A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.


Subject(s)
DNA Glycosylases , DNA/metabolism , Escherichia coli/enzymology , N-Glycosyl Hydrolases/metabolism , Uracil/pharmacology , Base Sequence , Binding Sites , Catalysis , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/genetics , Protein Binding , Substrate Specificity , Uracil-DNA Glycosidase
16.
J Biol Chem ; 273(21): 13170-6, 1998 May 22.
Article in English | MEDLINE | ID: mdl-9582358

ABSTRACT

We have previously developed an in vitro system that allows quantitative evaluation of the fidelity of transcription during synthesis on a natural template in the presence of all four nucleotides. Here, we have employed this system using a TAA ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase past an adenine residue adducted at the N6-position with (-)-anti-trans- or (+)-anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full-length run-off transcripts. The extent of bypass was found to be 32% for the (-)-anti-trans-isomer and 18% for the (+)-anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass synthesis of the (-)-anti-trans-isomer was elevated 11,000-fold and that of the (+)-anti-trans-isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template, three- and six-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell.


Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/chemical synthesis , Bacteriophage T7/enzymology , DNA Adducts/chemical synthesis , DNA-Directed RNA Polymerases/metabolism , Mutagens/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/chemistry , Base Sequence , DNA Adducts/chemistry , Isomerism , Templates, Genetic , Transcription, Genetic , Viral Proteins
17.
J Biol Chem ; 272(34): 21408-19, 1997 Aug 22.
Article in English | MEDLINE | ID: mdl-9261156

ABSTRACT

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex. Seven mutant Ugi proteins (E20I, E27A, E28L, E30L, E31L, D61G, and E78V) were created to assess the role of various negatively charged residues in the binding mechanism. Each mutant Ugi protein was purified and characterized with respect to inhibitor activity and Ung binding properties relative to the wild type Ugi. Analysis of the Ugi protein solution structures by nuclear magnetic resonance indicated that the mutant Ugi proteins were folded into the same general conformation as wild type Ugi. All seven of the Ugi proteins were capable of forming a Ung.Ugi complex but varied considerably in their individual ability to inhibit Ung activity. Like the wild type Ugi, five of the mutants formed an irreversible complex with Ung; however, the binding of Ugi E20I and E28L to Ung was shown to be reversible. The tertiary structure of [13C,15N]Ugi in complex with Ung was determined by solution state multi-dimensional nuclear magnetic resonance and compared with the unbound Ugi structure. Structural and functional analysis of these proteins have elucidated the two-step mechanism involved in Ung.Ugi association and irreversible complex formation.


Subject(s)
Bacillus Phages/enzymology , DNA Glycosylases , Escherichia coli/enzymology , N-Glycosyl Hydrolases/metabolism , Viral Proteins/chemistry , Binding, Competitive , Escherichia coli/genetics , Genes, Viral , Magnetic Resonance Spectroscopy , Models, Molecular , Mutagenesis, Site-Directed , Point Mutation , Protein Binding , Protein Structure, Tertiary , Structure-Activity Relationship , Uracil-DNA Glycosidase , Viral Proteins/metabolism , Viral Structural Proteins/genetics
18.
Cancer Res ; 57(14): 2956-60, 1997 Jul 15.
Article in English | MEDLINE | ID: mdl-9230208

ABSTRACT

Werner syndrome (WS) is an autosomal recessive disease, the phenotype of which is a caricature of premature aging. WS cells and cell lines display several types of genetic instability, and WS patients have an increased risk of developing cancer. The WS locus (WRN) encodes a protein that shows significant sequence homology to the RecQ family of DNA helicases. Because a DNA helicase may function in DNA mismatch repair, we examined extracts of WS cell lines for mismatch repair activity. Extracts from four different WS lymphoblastoid cell lines containing different WRN mutations and from three within-pedigree control cell lines were all proficient in mismatch repair. In marked contrast, extracts from three independent WS fibroblastoid cell lines were deficient in repair of base-base and insertion/deletion mismatches. Extracts of one of these lines restored activity to extracts of mismatch repair-deficient tumor cells with defined mutations in hMSH2, hMSH3, hMSH6, hMLH1, or hPMS2. This suggests that the WRN mutation in this fibroblast line is not a dominant negative inhibitor of mismatch repair activity and that the repair defect does not reside in these five known mismatch repair genes. Defective mismatch repair in fibroblastoid but not lymphoblastoid cells is consistent with the possibility that WRN protein could have a cell type- and/or tissue-specific role in mismatch repair. Alternatively, a mutation in WRN could predispose cells to mutations in other genes required for mismatch repair activity, at least one of which could be an unknown gene.


Subject(s)
DNA Helicases/physiology , DNA Repair , Werner Syndrome/genetics , Cell Line , Exodeoxyribonucleases , Genetic Complementation Test , Humans , RecQ Helicases , Werner Syndrome Helicase
19.
J Clin Psychol ; 52(5): 535-41, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8877688

ABSTRACT

The relation between childhood molestation and current adjustment, as measured by the Brief Symptom Inventory (BSI), was examined among women college students. Results suggest that the normative data available for the BSI are inappropriate for interpreting the performance of women college students and, particularly, students who have survived sexual abuse.


Subject(s)
Child Abuse, Sexual/psychology , Psychometrics/standards , Severity of Illness Index , Students/psychology , Survivors/psychology , Women/psychology , Adolescent , Adult , Female , Humans , Sampling Studies , Sick Role , Stress, Psychological/diagnosis
20.
J Biol Chem ; 270(28): 16840-7, 1995 Jul 14.
Article in English | MEDLINE | ID: mdl-7622499

ABSTRACT

The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that inactivates uracil-DNA glycosylase from diverse organisms. The secondary structure of Ugi consists of five anti-parallel beta-strands and two alpha-helices (Balasubramanian, S., Beger, R.D., Bennett, S.E., Mosbaugh, D.W., and Bolton, P.H. (1995) J. Biol. Chem. 270, 296-303). The tertiary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance. The Ugi structure contains an area of highly negative electrostatic potential produced by the close proximity of a number of acidic residues. The unfavorable interactions between these acidic residues are apparently accommodated by the stability of the beta-strands. This negatively charged region is likely to play an important role in the binding of Ugi to uracil-DNA glycosylase.


Subject(s)
Bacillus subtilis/virology , Bacteriophages/chemistry , DNA Glycosylases , N-Glycosyl Hydrolases/antagonists & inhibitors , Protein Structure, Tertiary , Viral Proteins/chemistry , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Uracil-DNA Glycosidase
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