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Biomedical subjects

M F Goodman

Publications and source records attributed to M F Goodman.

At least 55 records · Page 3Linked to original sources
PubMed1998

DNA mismatch repair catalyzed by extracts of mitotic, postmitotic, and senescent Drosophila tissues and involvement of mei-9 gene function for full activity.

Extracts of Drosophila embryos and adults have been found to catalyze highly efficient DNA mismatch repair, as well as repair of 1- and 5-bp loops. For mispairs T.G and G.G, repair is nick dependent and is specific for the nicked strand of heteroduplex DNA. In contrast, repair of A.A, C.A, G.A, C.T, T.T, and C.C is not nick dependent, suggesting the presence of glycosylase activities. For nick-dependent repair, the specific activity of embryo extracts was similar to that of extracts derived from the entirely postmitotic cells of young and senescent adults. Thus, DNA mismatch repair activity is expressed in Drosophila cells during both development and aging, suggesting that there may be a function or requirement for mismatch repair throughout the Drosophila life span. Nick-dependent repair was reduced in extracts of animals mutant for the mei-9 gene. mei-9 has been shown to be required in vivo for certain types of DNA mismatch repair, nucleotide excision repair (NER), and meiotic crossing over and is the Drosophila homolog of the yeast NER gene rad1.

Adenine↗
PubMed1997

DNA replication and postreplication mismatch repair in cell-free extracts from cultured human neuroblastoma and fibroblast cells.

DNA synthesis and postreplication mismatch repair were measured in vitro using cell-free extracts from cultured human SY5Y neuroblastoma and WI38 fibroblast cells in different growth states. All extracts, including differentiated SY5Y and quiescent WI38 fibroblasts, catalyzed SV40 origin-dependent DNA synthesis, totally dependent on SV40 T-antigen. Thus, although differentiated neuroblastoma and quiescent fibroblasts cells were essentially nondividing, their extracts were competent for DNA replication using DNA polymerases delta, alpha, and possibly epsilon, with proliferating cell nuclear antigen. Nonreplicative DNA synthesis and lesion bypass by either alpha- or beta-polymerases were detected independently in extracts using primed or gapped single-stranded DNA templates. Long-patch postreplication mismatch repair was measured for the first time in neuroblastoma cell-free extracts. Extracts from subconfluent and high-density SY5Y cells catalyzed postreplication mismatch repair with efficiencies comparable to those of HeLa cell extracts. No significant differences were observed in repair between SY5Y differentiated and undifferentiated cell extracts. Mismatch repair efficiencies were threefold lower in extracts from subconfluent WI38 cells, and repair in WI38 quiescent cells was fourfold less than in subconfluent cells, suggesting that mismatch repair may be regulated. The spectrum of mismatch repair in SY5Y extracts closely resembled the mismatch removal specificities of HeLa extracts: T . G and G . G mismatches were repaired most efficiently; C . A, A . A, A . G and a five-base loop were repaired with intermediate efficiency; repair of G . A, C . C, and T . T mismatches was extremely inefficient.

Antineoplastic Agents↗
PubMed1997

Fidelity of Escherichia coli DNA polymerase III holoenzyme. The effects of beta, gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies.

The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of beta, gamma processivity and epsilon proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies finc = 5.6 x 10(-6), 4.2 x 10(-7), and 7 x 10(-7), respectively. Elevated dGMP.G and dTMP.G misincorporation efficiencies of 3.2 x 10(-5) and 5.8 x 10(-4), attributed to a "dNTP-stabilized" DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct "rescue" nucleotide. As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide. Compared with pol III holoenzyme, the alpha holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP.G, dTMP.G, and dAMP.G mispairs. Both the beta, gamma complex and the downstream nucleotide have little effect on the fidelity of catalytic alpha subunit. An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the "Appendix" (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem. 272, 27931-27935 (accompanying paper)).

Catalysis↗
PubMed1997

The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II. Characterization of Pol II purified from a polB mutant.

Escherichia coli DNA polymerase II (Pol II) is a member of the group B, "alpha-like" family of DNA polymerases. Pol II is encoded by the damage-inducible dinA gene and exhibits SOS induction under the control of Lex A repressor. The polB gene was originally designated as the structural gene for Pol II based on the absence of detectable Pol II activity in cell lysates prepared from a strain containing the mutant polB100 allele. Because polB and dinA mapped at different chromosomal locations, it remained an open question whether polB, in addition to lexA, might be involved in regulating the expression of Pol II. We have cloned and sequenced the polB100 mutant allele, including adjacent surrounding sequences, and have expressed the mutant dinA gene from Pol B100 on a high copy number plasmid. Our sequence data reveal that polB and dinA represent the same gene and that the original transduction mapping of polB was inaccurate. We purified the mutant Pol B100 polymerase and show that it retains 5 to 10% of the wild-type level of polymerase activity. The Pol B100 mutation, Gly401 --> Asp401, is not located within any of the five conserved domains that define group B polymerases. Pol B100 retains a wild-type level of 3' --> 5' exonuclease activity. We suggest that the normal level of exonucleolytic proofreading associated with the mutant Pol B100 enzyme may explain the repeated failures, over the past two decades, to detect phenotypes in polB mutant strains.

Bacterial Proteins↗
PubMed1997

Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA synthesis in vivo.

We have investigated a role for Escherichia coli DNA polymerase II (Pol II) in copying chromosomal and episomal DNA in dividing cells in vivo. Forward mutation frequencies and rates were measured at two chromosomal loci, rpoB and gyrA, and base substitution and frameshift mutation frequencies were measured on an F'(lacZ) episome. To amplify any differences in polymerase error rates, methyl-directed mismatch repair was inactivated. When wild-type Pol II (polB+) was replaced on the chromosome by a proofreading-defective Pol II exo- (polBex1), there was a significant increase in mutation frequencies to rifampicin resistance (RifR) (rpoB) and nalidixic acid resistance (NalR) (gyrA). This increased mutagenesis occurred in the presence of an antimutator allele of E. coli DNA polymerase III (Pol III) (dnaE915), but not in the presence of wild-type Pol III (dnaE+), suggesting that Pol II can compete effectively with DnaE915 but not with DnaE+. Sequencing the RifR mutants revealed a G --> A hot spot highly specific to Pol II exo-. Pol II exo- caused a significant increase in the frequency of base substitution and frameshift mutations on F' episomes, even in dnaE+ cells, suggesting that Pol II is able to compete with Pol III for DNA synthesis on F episomes.

Anti-Bacterial Agents↗
PubMed1997

Abasic translesion synthesis by DNA polymerase beta violates the "A-rule". Novel types of nucleotide incorporation by human DNA polymerase beta at an abasic lesion in different sequence contexts.

The "A-rule" reflects the preferred incorporation of dAMP opposite abasic lesions in Escherichia coli in vivo. DNA polymerases (pol) from procaryotic and eucaryotic organisms incorporate nucleotides opposite abasic lesions in accordance with the A-rule. However, recent in vivo data demonstrate that A is not preferentially incorporated opposite abasic lesions in eucaryotes. Purified human DNA polymerases beta and alpha are used to measure the specificity of nucleotide incorporation at a site-directed tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream and downstream bases adjacent to the lesion. Extension past the lesion is measured in 4 sequence contexts, varying the downstream template base. Pol alpha strongly favors incorporation of dAMP directly opposite the lesion. In marked contrast, pol beta violates the A-rule for incorporation directly opposite the lesion. In addition to incorporation taking place directly opposite the lesion, we also analyze misalignment incorporation directed by a template base downstream from the lesion. Lesion bypass by pol beta occurs predominantly by "skipping over" the lesion, by insertion of a nucleotide complementary to an adjacent downstream template site. Misalignment incorporation for pol beta occurs by a novel "dNTP-stabilized" mechanism resulting in both deletion and base substitution errors. In contrast, pol alpha shows no propensity for this type of synthesis. The misaligned DNA structures generated during dNTP-stabilized lesion bypass do not conform to misaligned structures reported previously.

Base Sequence↗
PubMed1997

Relevance of 'adaptive' mutations arising in non-dividing cells of microorganisms to age-related changes in mutant phenotypes of neurons.

Brattleboro rats do not produce vasopressin (VP) because of a germ-line single-base deletion (di) that causes a frame shift downstream from the VP sequences and a loss of a stop codon. The resulting frame-shifted peptide precursor does not enter the secretory pathway in hypothalamic neurons, thereby blocking the neurosecretion of VP and other peptides. Yet, from birth onwards, a subpopulation of neurons in di/di rats slowly accumulates revertant cells with a hemizygous wild-type phenotype. Because the rate of reversion during aging is slowed by vasopressin infusion, it is of interest to consider these phenomena in relation to recent observations on 'adaptive' mutations in single cell bacteria and yeast that enable reversion of mutations that blocked cell division under conditions of nutrient deficits. In considering mechanisms that could produce revertant phenotypes in non-dividing cells of both pro- and eukaryotes, we note the pertinence of transcription-coupled repair and SOS 'error-prone' repair.

Adaptation, Physiological↗
PubMed1996

Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA.

A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.

Adenosine Triphosphate↗
PubMed1996

Base analog and neighboring base effects on substrate specificity of recombinant human G:T mismatch-specific thymine DNA-glycosylase.

We studied the substrate specificity of the human G:T mismatch-specific thymine glycosylase that initiates the repair of G:T and G:U base mismatches to G:C base pairs. Such mismatches arise when 5-methylcytosine or cytosine deaminate spontaneously (and hydrolytically) in DNA. Substrates were 45-bp DNA heteroduplexes that bore single G:T, m6G:T, 2,6-diaminopurine:T, 2-amino-6-(methylamino)-purine:T, 2-aminopurine:T, and G:m4T mispairs. The bases 5' to the poorly matched G were altered in selected G:T substrates to yield mispairs in four different contexts, ApG, CpG, GpG, and TpG. The recombinant thymine glycosylase was incubated with the 45-bp DNA substrates, each labeled at the 5'-terminus of the strand containing the mismatched T. The DNAs were then treated with 0.1 N NaOH to catalyze phosphodiester bond breakage at the newly-generated AP sites, and the products were analyzed on DNA sequencing gels. As indicated by the amounts of the 20-nt incision product, the removal of the thymine base by the enzyme increased linearly between 0 and 40 min at which time the generation of product from all substrates ceased, probably because of enzyme inactivation. The rate of incision was greatest (0.7 fmol/min) with DNA containing the G:T mispair followed by the DNA containing the m6G:T mispair (0.38 fmol/min) and the DNA with the 2-amino-6-(methylamino)purine:T mispair (0.15 fmol/ min); the extent of reaction was 90%, 40%, and 20% respectively. By contrast to previous findings with cell-free extracts, DNA substrates containing 2,6-diaminopurine:T, 2-aminopurine:T, and G:m4T mispairs were not incised (< 2%). The amount of incision of the 45-bp DNA substrates containing G:T mispairs in the CpG context was 3-12-fold greater than in the TpG, GpG, and ApG contexts.

5-Methylcytosine↗
PubMed1996

Spectroscopic and calorimetric characterizations of DNA duplexes containing 2-aminopurine.

The base analog 2-aminopurine (AP) strongly promotes A.T to G.C and G.C to A.T transitions in bacteria and bacteriophage. During DNA replication, the primary mutagenic event involves formation of a heteroduplex with an AP.C site at a much higher frequency than formation of the corresponding heteroduplex with an A.C site. It is not known if AP-induced mutagenesis correlates with differences in the thermodynamic properties of an AP.C versus an A.C site, or whether interactions involving DNA polymerases are controlling. To address this specific question, and more generally to characterize AP-containing duplexes, we have used a combination of spectroscopic and calorimetric techniques to determine the thermodynamic properties of six 11-mer duplexes. The sequences of these duplexes are identical except for the identity of the variable central base pair which can be either A.T, A.C, AP.T, AP.C, AP.A, or AP.G, and which we use to designate each duplex. Analyses and interpretation of the optically and calorimetrically derived thermal and thermodynamic data on these six duplexes reveal the relative stabilizing influence of the central base pairs to be A.T > AP.T > AP.C > AP.A > AP.G > A.C, with the AP.C-containing duplex being significantly more stable than the A.C-containing duplex. In the aggregate, our results suggest that during incorporation, base pair discrimination by DNA polymerases is influenced, in part, by differences in the thermodynamic stabilities of the newly formed base pairs.

2-Aminopurine↗
PubMed1996

Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases.

Expansions of trinucleotide repeats in DNA, a novel source of mutations associated with human disease, may arise by DNA replication slippage initiated by hairpin folding of primer or template strands containing such repeats. To evaluate the stability of single-strand folding by repeating triplets of DNA bases, thermal melting profiles of (CAG)10, (CTG)10, (GAC)10 and (GTC)10 strands are determined at low and physiological salt concentrations, and measurements of melting temperature and enthalpy change are made in each case. Comparisons are made to strands with three times as many repeats, (CAG)30 and (CTG)30. Evidence is presented for stable intrastrand folding by the CAG/CTG class of triplet repeats. Relative to the GAC/GTC class not associated with disease, the order of folding stability is found to be CTG > GAC approximately = CAG > GTC for 10 repeats. Surprisingly, the folds formed by 30 repeats of CTG or CAG have no higher melting temperature and are only 40% more stable in free energy than those formed by 10 repeats. This finding suggests that triplet expansions with higher repeat number may result from the formation of more folded structures with similar stability rather than fewer but longer folds of greater stability.

Base Sequence↗
PubMed1996

Purification of a soluble UmuD'C complex from Escherichia coli. Cooperative binding of UmuD'C to single-stranded DNA.

The Escherichia coli UmuD' and UmuC proteins play essential roles in SOS-induced mutagenesis. Previous studies investigating the molecular mechanisms of mutagenesis have been hindered by the lack of availability of a soluble UmuC protein. We report the extensive purification of a soluble UmuD'C complex and its interactions with DNA. The molecular mass of the complex is estimated to be 70 kDa, suggesting that the complex consists of one UmuC (46 kDa) and two UmuD' (12 kDa) molecules. In contrast to its inability to bind to double-stranded DNA, UmuD'C binds cooperatively to single-stranded DNA as measured by agarose gel electrophoresis and confirmed by steady-state fluorescence depolarization. A Hill coefficient, n = 3, characterizes the binding of UmuD'C to M13 DNA and to a 600 nucleotide DNA oligomer, suggesting that at least three protein complexes may interact cooperatively when binding to DNA. The apparent equilibrium binding constant of UmuD'C to single-stranded DNA is approximately 300 nM. Binding of the complex to a short, 80 nucleotide, DNA oligonucleotide was detectable by fluorescence depolarization, but it did not appear to be cooperative. Binding of UmuD'C to single-stranded M13 DNA causes an acceleration of the protein-DNA complex, suggesting that the longer DNA may undergo compaction. The UmuD'C complex associates with RecA-coated DNA, and the UmuD'C complex remains bound to DNA in the presence of RecA.

Amino Acid Sequence↗
PubMed1996

NMR study of the conformation of the 2-aminopurine:cytosine mismatch in DNA.

DNA polymerase makes errors by misincorporating natural DNA bases and base analogs. Because of the wide variety of possible mismatches and the varying efficiency with which they are repaired, structural studies are necessary to understand in detail how these mispairs differ and can be distinguished from standard Watson-Crick base pairs. 2-Aminopurine (AP) is a highly mutagenic base analog. The objective of this study was to determine the geometry of the AP x C mispair in DNA at neutral pH. Although several studies have focused on the AP x C mispair in DNA, there is not as of yet consensus on its structure. At least four models have been proposed for this mispair. Through the use of NMR spectroscopy with selective 15N-labeling of exocyclic amino nitrogens on bases of interest, we are able to resolve ambiguities in previous studies. We find here that, in two different DNA sequences, the AP x C mispair at neutral and high pH is in a wobble geometry. The structure and stability of this base mispair is dependent upon the local base sequence.

2-Aminopurine↗
PubMed1995

Proofreading-defective DNA polymerase II increases adaptive mutation in Escherichia coli.

The role of Escherichia coli DNA polymerase (Pol) II in producing or avoiding mutations was investigated by replacing the chromosomal Pol II gene (polB+) by a gene encoding an exonuclease-deficient mutant Pol II (polBex1). The polBex1 allele increased adaptive mutations on an episome in nondividing cells under lactose selection. The presence of a Pol III antimutator allele (dnaE915) reduced adaptive mutations in both polB+ cells and cells deleted for polB (polB delta 1) to below the wild-type level, suggesting that both Pol II and Pol III are synthesizing episomal DNA in nondividing cells but that in wild-type cells Pol III generates the adaptive mutations. The adaptive mutations were mainly -1 frame-shifts occurring in short homopolymeric runs and were similar in wild-type, polB delta 1, and polBex1 strains. Mutations produced by both Pol III and Pol II ex1 were corrected by the mutHLS mismatch repair system.

Acclimatization↗
PubMed1995

Purification and properties of wild-type and exonuclease-deficient DNA polymerase II from Escherichia coli.

Wild-type DNA polymerase II (pol II) and an exonuclease-deficient pol II mutant (D155A/E157A) have been overexpressed and purified in high yield from Escherichia coli. Wild-type pol II exhibits a high proofreading 3'-exonuclease to polymerase ratio, similar in magnitude to that observed for bacteriophage T4 DNA polymerase. While copying a 250-nucleotide region of the lacZ alpha gene, the fidelity of wild-type pol II is high, with error rates for single-base substitution and frameshift errors being < or = 10(-6). In contrast, the pol II exonuclease-deficient mutant generated a variety of base substitution and single base frameshift errors, as well as deletions between both perfect and imperfect directly repeated sequences separated by a few to hundreds of nucleotides. Error rates for the pol II exonuclease-deficient mutant were from > or = 13- to > or = 240-fold higher than for wild-type pol II, depending on the type of error considered. These data suggest that from 90 to > 99% of base substitutions, frameshifts, and large deletions are efficiently proofread by the enzyme. The results of these experiments together with recent in vivo studies suggest an important role for pol II in the fidelity of DNA synthesis in cells.

Base Sequence↗