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Biomedical subjects

B S Strauss

Publications and source records attributed to B S Strauss.

At least 37 records · Page 2Linked to original sources
PubMed1990

Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA.

N-(Deoxyguanosin-8-yl)-2-(acetylamino)fluorene (AAF-G) adducts in the DNA of bacteriophage M13 can be converted to N-(deoxyguanosin-8-yl)-2-aminofluorene (AF-G) adducts in situ by treatment with 1.0 M NaOH for 45 min at room temperature. The conversion is accompanied by a dramatic increase in the transfection activity of the samples which is correlated with the measured deacetylation of the acetylaminofluorene adduct. The pair of substrates (AAF-G/AF-G) with adducts at identical places in the DNA has been used to study bypass synthesis catalyzed by T7 DNA polymerase, an altered T7 DNA polymerase from which the 3'----5' exonuclease has been genetically removed by an 84 nucleotide deletion (Sequenase 2), T4 DNA polymerase and Escherichia coli DNA polymerase I. All polymerases appear blocked at acetylaminofluorene lesions. Sequenase 2 is apparently able to add nucleotides opposite the acetylaminofluorene lesion but is unable to catalyze further elongation. T7 DNA polymerase, including thioredoxin and with an active 3'----5' exonuclease, is unable to bypass aminofluorene adducts, whereas Sequenase 2 bypasses the lesions readily. The data support the view that the elongation step is rate limiting in synthesis past lesions and that low 3'----5' exonuclease activity allows the priming nucleotide opposite the altered template site to remain in position long enough for elongation past particular adducts.

Acetoxyacetylaminofluorene↗
PubMed1989

In vitro mutagenesis and SOS repair.

SOS repair in Escherichia coli (E. coli) utilizes inducible gene products to "fix" mutations in the genome. It has been supposed that an "error prone" system is induced which makes errors in the course of bypassing lesions. An alternative model has been proposed by Bridges and Woodgate which suggests that it is the elongation step rather than the insertion opposite a damaged site which is critical in SOS mutagenesis. A variety of in vitro evidence supports this more recent model. DNA polymerases can be shown to insert bases opposite non-instructional lesions in DNA. It can be demonstrated that it is the elongation rather than the insertion step which is rate limiting in such in vitro reactions. An in vitro mutagenesis system can be devised using a processive polymerase with a reduced 3'----5' exonuclease activity. This system mimics the mutagenic specificity seen in vivo.

DNA Repair↗
PubMed1987

Influence of template strandedness on in vitro replication of mutagen-damaged DNA.

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Predictors of outcome in schizophrenia: the process-reactive dimension.

Premorbid social-sexual competence and the process-reactive dimension, as assessed by the Phillips Scale, were investigated as predictors of posthospital adjustment in a prospective study of two samples of 141 young, early phase schizophrenics (a private hospital sample and a state hospital sample). The Phillips Scale predicted subsequent outcome at significant levels for one of the two samples of DSM-II schizophrenics (a "broad" construct of schizophrenia). It did not predict outcome as well for DSM-III schizophrenics (a "narrow" construct of schizophrenia). Mixed results emerged from separate analyses of first-admission schizophrenics only and for male schizophrenics only. The data indicated that marital status accounts for some of the positive relationship between the Phillips Scale and later outcome. A more focused measure of prehospital social adjustment successfully predicted social functioning at followup for both DSM-II and DSM-III schizophrenics (p less than .05). There were some significant and near-significant relationships, but overall results suggest that when the influence of chronicity and marital status is reduced, the relationship between the Phillips Scale and subsequent outcome is less robust than was once thought.

Adult↗
PubMed1985

Cautions in alternate-form presentation of aural test material: Speech Sounds Perception Test.

86 college students were administered the standard Speech Sounds Perception Test and an alternate recording. Although reliability between the two recordings was moderate (r = .68) when the standard was given first, but only .36 when the alternate form was given first, differences in performance were consistent with differences in attack and decay found in voice-print tracings. Results suggest caution in using an alternate form of a neuro-psychological test, particularly one using auditory stimuli, until cross-validation confirms the reliability coefficient and the level of performance.

Adult↗
PubMed1985

Translesion DNA synthesis: polymerase response to altered nucleotides.

A system for the determination of the specificity of incorporation opposite lesions during DNA synthesis past damaged bases has been used as a model of mutation. The system, based on the dideoxynucleotide sequence method, uses lesions in the template strand as chain terminators. As a first approximation, such lesions constitute non-instructive sites in the DNA. DNA synthesis catalysed by bacteriophage T4 DNA polymerase terminates one nucleotide 3' to the lesion on the template strand. With other polymerases, synthesis may terminate opposite the lesion. The details of termination site depend on the enzyme, the metal ion (Mg2+ or Mn2+), the lesion and the particular nucleotide sequence. The sequence effect for termination on normal templates has two components, one ascribable to secondary structure, the other intrinsic to the sequence itself. DNA molecules terminated before lesions may be used as substrates in 'second stage' reactions in which elongation can be detected on the addition of particular dNTPs (deoxynucleoside triphosphates) to a reaction mixture. The specificity of elongation depends on the polymerase, on the activity of the 3'--greater than 5' editing nuclease and on the particular lesion. DNA polymerases have a preference for the addition of purines, particularly adenine, opposite non-instructional sites. This preference suggests an explanation for the specificity of base substitution mutations: treatments that produce non-instructional sites from purines will lead to transversions, treatments that affect pyrimidines lead to transitions. Even though a base is added opposite a lesion, further elongation may be rate limiting. Whether or not elongation occurs is dependent on the sequence 5' to the lesion on the template strand. The interactions of the factors affecting bypass: polymerase, lesion and sequence, may well result in an idiosyncratic behaviour for each mutable site.

Animals↗
PubMed1985

In vitro models of mutagenesis.

The bypass of lesions in DNA with insertion of nucleotides opposite damaged bases has been studied as a model for mutagenesis in an in vitro system. Lesions introduced by dimethyl sulfate at adenines and by ultraviolet light at pyrimidine dimers act as termination sites on both double- and single-stranded DNA templates. Base selection opposite noninformational lesions is, in part, a property of the polymerases: different polymerases have different selectivities although all polymerases tested seem to prefer purines. The ability to insert "incorrect" bases is determined in part by the sequence 5' to the lesion on the template strand. The hypothesis that damaged purines tend to result in transversions can be applied to published data on activation of the c-ras oncogene.

Base Sequence↗
PubMed1984

A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts.

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase alpha from both calf thymus and human lymphoma cells and DNA polymerase beta from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG lesions when Mg2+ is the divalent cation. Substitution of Mn2+ for Mg2+ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase alpha inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase beta is specific, inserting mainly C even in the presence of Mn2+. The Km for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn2+ is about 0.5 mM. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg2+ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn2+. dNTP alpha S and recA protein increase only the insertion of C. We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.

2-Acetylaminofluorene↗
PubMed1984

The interaction of u.v.- and methyl methanesulfonate-induced DNA repair synthesis: a role for poly(ADP-ribose)?

Treatment of a human lymphoblastoid line ( GM606 ) with methyl methanesulfonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initially stimulates and then drastically inhibits repair synthesis. This inhibition is overcome and repair appears to be stimulated by the poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide (3-AmB). The repair activity of older cultures is much diminished compared to those rapidly dividing, but the 3-AmB effect is seen in both 2- and 7-day cultures. The action of 3-AmB is very rapid and its removal results in a diminution of repair synthesis within 30 min. U.v.-induced repair synthesis was not affected by 3-AmB. However, the level of u.v.-induced repair synthesis was drastically reduced by treatment of cells with MMS. This MMS inhibition of u.v.-induced repair synthesis was completely counteracted by the presence of 3-AmB. Insofar as the effective action of 3-AmB is on poly(ADP-ribose) synthesis, the data suggest that activation of poly(ADP-ribose) synthesis by alkylation of DNA reduces the NAD and therefore the ATP level in the lymphoblastoid cells used. Reduced ATP levels result in a lessened ability to carry out u.v.-induced repair synthesis.

Cell Line↗
PubMed1983

In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation.

A variety of DNA polymerases, synthesizing in vitro on an UV-irradiated phi X174 DNA template, terminate synthesis one nucleotide before the 3' pyrimidines of putative dimers on the template. We have devised a system using Escherichia coli DNA polymerase I (Klenow fragment) that can synthesize past at least some of these dimers. The bypass is carried out in a multistep process--first, the incorporation of nucleotides opposite the pyrimidines in the dimer and, then, the addition of nucleotides complementary to the bases distal to the dimer. The insertion of a nucleotide opposite the first (3') pyrimidine of a putative dimer in the presence of Mn2+ occurs in a concentration-dependent fashion with a 3- to 4-fold preference for purine nucleotides over pyrimidine nucleotides. In the presence of Mg2+, insertion is less frequent. Correlation of these results with in vivo mutation data suggests a role for the polymerase in determining the spectrum of base substitution mutagenesis in SOS induced cells.

Base Sequence↗
PubMed1983

O6-Methylguanine removal by competent and incompetent human lymphoblastoid lines from the same male individual.

Two cell lines, one proficient (Mex+) and one deficient (Mex-) in the ability to remove O6-methylguanine, have been isolated by Epstein-Barr virus-mediated transformation of a single blood sample obtained from a normal human male. Extracts of untreated cells differ in their O6-methylguanine transferase (methyl acceptor protein) activity. Although both lines arise from the same individual, they show great difference in their sensitivities to the toxic action of N-methyl-N'-nitro-N-nitrosoguanidine. Chromosome counts of the strains reveal a modal number of 46 for both. Neither X-inactivation nor a gross abnormality in chromosome number can be the cause of the difference between the two lines.

Adenine↗
PubMed1982

In vitro replication of mutagen-damaged DNA: sites of termination.

We have examined the effect of DNA lesions, which in vivo are potentially mutagenic, on in vitro DNA synthesis carried out by a number of purified DNA polymerases using a 0X174 template. Both acetyl aminofluorene (AAF) adducts and UV-induced pyrimidine dimers are blocks to elongation by DNA polymerases. On UV-irradiated DNA templates synthesis terminates one nucleotide before the sites of pyrimidine dimers with all of the enzymes tested: Pol I and Pol III holoenzyme from Escherichia coli, T4 DNA polymerase, avian myeloblastosis virus reverse transcriptase and a mammalian DNA polymerase alpha. With AAF, which reacts at the C-8 position of guanine, differences are observed between the above enzymes, with the latter two inserting a nucleotide opposite the site of the lesion. Substitution of Mn2+ for Mg2+ as the cation in the Pol I reactions causes changes in the termination pattern on both UV-irradiated and AAF-reacted templates. The significance of these results to the process of inducible error-prone repair and the possible bypass of lesions in the DNA is discussed.

Bacteriophage phi X 174↗
PubMed1982

Effect of acetylated and deacetylated 2-aminofluorene adducts on in vitro DNA synthesis.

We have constructed primed phi X174 DNA templates containing either acetylated or deacetylated aminofluorene adducts at the C-8 position of guanine. T4 DNA polymerase terminates synthesis one nucleotide before the acetylated adducts but incorporates an additional nucleotide opposite the deacetylated guanylaminofluorene. These observations can be explained by the known preferred conformations of the acetylated and deacetylated guanosinylaminofluorene nucleosides--the former favoring the syn conformation (so that in DNA the guanine is displaced from the helix by the fluorene ring) and the latter preferring the anti conformation (which allows normal base pairing of the guanine with cytosine). A similar differentiation between the two adducts was found with Escherichia coli DNA polymerase I. In contrast, avian myeloblastosis virus (AMV) reverse transcriptase, which terminated with a nucleotide inserted opposite the acetylated adducts, was less able to do so at the deacetylated adducts. The nucleoside incorporated by AMV reverse transcriptase opposite the acetylated adduct was exclusively cytidine, which suggests regular base pairing with the reacted guanosine nucleoside in the anti conformation; however, synthesis was completely blocked and unable to continue beyond this point. The differences between the termination patterns of the prokaryotic enzymes and AMV reverse transcriptase indicates that specific properties of a replicating polymerase can influence the conformation of a reacted nucleoside in the DNA, thus altering its recognition and possibly its mutagenic activity.

2-Acetylaminofluorene↗
PubMed1981

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

In vitro DNA synthesis on a phi X174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol I) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4 DNA polymerase, a polymerase alpha from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3' leads to 5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase alpha preparations. On N-acetylaminofluorene templates, pol I, pol III, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase alpha-catalyzed reactions terminate at the site of the lesion. Substitution of Mn2+ for Mg2+ changes the pattern of pol I-catalyzed termination sites. The data suggest that termination is a complicated process that does not depend exclusively on the 3' leads to 5' exonuclease activity associated with many polymerases.

2-Acetylaminofluorene↗
PubMed1980

Apurinic/apyrimidinic endonuclease sensitive sites as intermediates in the in vitro degradation of deoxyribonucleic acid by neocarzinostatin.

Neocarzinostatin (NCS) induces alkali-labile sites in DNA which are stabilized by NaBH4 reduction. The stabilized sites are sensitive to an AP endonuclease from human lymphoma cells. NCS-induced degradation of supercoiled Col E1 DNA proceeds in stepwise fashion with apurinic/apyrimidinic (AP) sites as intermediates. Degradation is increased when reaction occurs in the presence of AP endonuclease, and DNA reacted with NCS can be shown to have numerous AP endonuclease sensitive sites.

Antibiotics, Antineoplastic↗
PubMed1980

Repair of neocarzinostatin-induced deoxyribonucleic acid damage in human lymphoblastoid cells: possible involvement of apurinic/apyrimidinic sites as intermediates.

Neocarzinostatin (NCS) induces repair in a xeroderma pigmentosum lymphoblastoid line deficient in the ability to repair DNA damage induced with (acetoxyacetyl-amino)fluorene. Repair was demonstrated by the induction of repair synthesis and by the disappearance of NCS-induced single-strand breaks and/or alkaline-labile sites in DNA. Estimation of NCS-induced repair patch size, based on the density shift induced in DNA by extensive shear after incubation of treated cells in medium with bromodeoxyuridine or by calculation from the extent of restoration of DNA sedimentation profiles in alkaline sucrose gradients and the amount of repair synthesis measured by the BND cellulose method, indicated that only a few nucleotides were inserted per repaired region. NCS-treated bacteriophage T7 DNA requires incubation with alkaline phosphatase to make it a substrate for DNA polymerase I. NCS-reacted T7 DNA, even after phosphatase treatment, is not a substrate for a DNA polymerase alpha obtained from human lymphoma cells. NCS-treated T7 DNA did serve as a substrate for the DNA polymerase alpha when incubated with an apurinic/apyrimidinic (AP) endonuclease with associated 5'-3'-exonuclease activity. The results suggest that NCS-induced AP sites could be intermediates for the in vivo repair synthesis.

Antibiotics, Antineoplastic↗