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Myron F Goodman

Publications and source records attributed to Myron F Goodman.

At least 37 records · Page 2Linked to original sources
PubMed2004

Effects of the C4'-oxidized abasic site on replication in Escherichia coli. An unusually large deletion is induced by a small lesion.

The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress by a variety of agents. For instance, the lesion accounts for approximately 40% of the DNA damage produced by the antitumor antibiotic bleomycin. The effect of C4-AP on DNA replication in Escherichia coli was determined using the restriction endonuclease and postlabeling (REAP) method. Three-nucleotide deletion products are the sole products observed following replication of plasmids containing C4-AP under SOS conditions in wild-type cells. Full-length products are formed in varying amounts depending upon the local sequence in wild-type cells under non-SOS-induced conditions. The "A-rule" is followed for the formation of substitution products. C4-AP is the first example of a DNA lesion that produces significant levels of three-nucleotide deletions in a variety of sequence contexts. Experiments carried out in cells lacking specific polymerases reveal that formation of three-nucleotide deletion products results from a coordinated effort involving pol II and pol IV. This is the first example in which these SOS inducible polymerases are shown to work in concert during lesion bypass. Three-nucleotide deletions are not observed during the replication of other abasic lesions, and are rarely produced by bulky adducts. The effect of C4-AP on DNA replication suggests a significant role for this lesion in the cytotoxicity of bleomycin. Formation of the C4-AP lesion may also be responsible for the formation of mutant proteins containing single-amino acid deletions that exhibit altered phenotypes.

Apurinic Acid↗
PubMed2004

A comprehensive comparison of DNA replication past 2-deoxyribose and its tetrahydrofuran analog in Escherichia coli.

Apurinic/apyrimidinic (AP) sites are alkali labile lesions that, when encountered during DNA replication, can block polymerases or potentially result in mutagenic events. Owing to the instability of 2-deoxyribose lesions (AP), a chemically stable tetrahydrofuran analog (F) is often used as a model of abasic sites. A comparison of the two lesions in Saccharomyces cerevisiae revealed that the model lesion and 2-deoxyribose have distinct in vivo effects. Comprehensive comparative analyses of F and AP have not been carried out in Escherichia coli. We conducted a side-by-side investigation of F and AP in E.coli to compare their biological effects and interactions with SOS polymerases. Both lesions were examined in SOS-induced and uninduced cells. Our studies reveal that in uninduced E.coli the effects of individual polymerases in the replication of plasmids containing F or AP are distinct. However, when cells are SOS-induced, the biological effects of F and AP are similar.

DNA Damage↗
PubMed2004

Biochemical analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase.

The synthesis of high affinity antibodies requires activation-induced cytidine deaminase (AID) to initiate somatic hypermutation and class-switch recombination. Here we investigate AID-catalyzed deamination of C --> U on single-stranded DNA and on actively transcribed closed circular double-stranded DNA. Mutations are initially favored at canonical WRC (W = A or T, R = A or G) somatic hypermutation hot spot motifs, but over time mutations at neighboring non-hot spot sites increase creating random clusters of mutated regions in a seemingly processive manner. N-terminal AID mutants R35E and R35E/R36D appear less processive and have altered mutational specificity compared with wild type AID. In contrast, a C-terminal deletion mutant defective in CSR in vivo closely resembles wild type AID. A mutational spectrum generated during transcription of closed circular double-stranded DNA indicates that wild type AID retains its specificity for WRC hot spot motifs within the confines of a moving transcription bubble while introducing clusters of multiple deaminations predominantly on the nontranscribed strand.

Amino Acid Motifs↗
PubMed2004

To slip or skip, visualizing frameshift mutation dynamics for error-prone DNA polymerases.

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) mu catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol mu and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol mu switches to dNTP-stabilized misalignment. pol beta generates -1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.

Catalysis↗
PubMed2004

Increased dNTP binding affinity reveals a nonprocessive role for Escherichia coli beta clamp with DNA polymerase IV.

Replication forks often stall at undamaged or damaged template sites in Escherichia coli. Subsequent resumption of DNA synthesis occurs by replacing DNA polymerase III, which is bound to DNA by the beta-sliding clamp, with one of three damage-induced DNA polymerases II, IV, or V. The principal role of the beta clamp is to tether the normally weakly bound polmerases to DNA thereby increasing their processivities. DNA polymerase IV binds dNTP substrates with about 10-fold lower affinity compared with the other E. coli polymerases, which if left unchecked could hinder its ability to synthesize DNA in vivo. Here we report a new property for the beta clamp, which when bound to DNA polymerase IV results in a large increase in dNTP binding affinity that concomitantly increases the efficiency of nucleotide incorporation at normal and transiently slipped mispaired primer/template ends. Primer-template DNA slippage resulting in single nucleotide deletions is a biological hallmark of DNA polymerase IV infidelity responsible for enhancing cell fitness in response to stress. We show that the increased DNA polymerase IV-dNTP binding affinity is an intrinsic property of the DNA polymerase IV-beta clamp interaction and not an indirect consequence of an increased binding of DNA polymerase IV to DNA.

Base Pair Mismatch↗
PubMed2004

Mutagenic effects of 2-deoxyribonolactone in Escherichia coli. An abasic lesion that disobeys the A-rule.

Abasic sites are often referred to as noninstructive lesions. The C1'-oxidized abasic site (2-deoxyribonolactone, L) is produced by several DNA damaging agents, including gamma-radiolysis and the neocarzinostatin chromophore (NCS). The effects of a C1'-oxidized abasic site incorporated at a defined site in single-stranded plasmid were examined in SOS polymerase-proficient and -deficient Escherichia coli. For comparison, experiments utilizing plasmids containing an abasic site (AP) were carried out side by side. In contrast to plasmid containing AP, dA and dG were incorporated most often when plasmid containing L was replicated. The ratio of dG:dA incorporation depended upon local sequence and varied from 0.9 to 2.2. High levels of translesion incorporation of dA are consistent with previous observations that treatment of DNA with the neocarzinostatin chromophore resulted in large amounts of G.C --> A.T transitions [Povirk and Goldberg (1986) Nucleic Acids Res. 14, 1417] and support the proposal that L is the source of these mutations. Both abasic lesions were 100% lethal in triple knockout cells lacking pol II, pol IV, and pol V. Analysis of translesion synthesis in repair-deficient cells revealed that pol V played a significant role in replication of L and AP. Significant levels of -1 frameshifts were formed in 5'-d(CL) sequences in the presence of pol V and were the exclusive product in pol V-deficient cells. Frameshift products were not formed when the nucleotide on the 5'-side of L was either dT or dG. Deleting pol II or pol IV had only modest effects on replication of L-containing plasmid but significantly decreased the amount of -1 frameshift product formed from an AP lesion. Experiments carried out side by side using otherwise identical plasmids containing an AP site illustrate the distinct properties of these two abasic lesions and that neither should be thought of as noninstructive.

Bacteriophage lambda↗
PubMed2004

In vitro effects of a C4'-oxidized abasic site on DNA polymerases.

Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites. The C4'-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents. This lesion accounts for approximately 40% of the DNA damage produced by bleomycin. The effect of a C4'-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3' --> 5' exonuclease activity. Both enzymes preferentially incorporated dA > dG >> dC, T opposite C4-AP. Neither enzyme is able to extend the primer past the lesion. Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites. Extension of the primer by alternative polymerases pol II, pol II exo(-), pol IV, and pol V was examined. Pol II exo(-) was most efficient. Qualitative translesion synthesis experiments showed that pol II exo(-) preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC. Thymidine incorporation opposite C4'-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template. These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the "A-rule". The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C --> T:A transversions in cells treated with bleomycin.

Carbon↗
PubMed2004

In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.

BACKGROUND: Clinical specimens are routinely fixed in 10% buffered formalin and paraffin embedded. Although DNA is commonly extracted from fixed tissues and amplified by PCR, the effects of formalin fixation are relatively unknown. Formalin fixation is known to impair PCR, presumably through damage that blocks polymerase elongation, but an insidious possibility is error prone translesion synthesis across sites of damage, producing in vitro artifactual mutations during PCR. METHODS: To better understand the consequences of fixation, DNA specimens extracted from fresh or fixed tissues were amplified with Taq DNA polymerase, and their PCR products were cloned and sequenced. RESULTS: Significantly more (3- to 4-fold) mutations were observed with fixed DNA specimens. The majority of mutations were transitions, predominantly at A:T base pairs, randomly distributed along the template. CONCLUSIONS: Formalin fixation appears to cause random base damage, which can be bridged during PCR by Taq DNA polymerase through error prone translesion synthesis. Fixed DNA is a damaged but "readable" template.

Journal Article↗
PubMed2004

Error-prone replication for better or worse.

Precise genome duplication requires accurate copying by DNA polymerases and the elimination of occasional mistakes by proofreading exonucleases and mismatch repair enzymes. The commonly held belief that 'if something is worth doing, then it's worth doing well' normally applies to DNA replication and repair, however, there are exceptions. This review describes elements that are crucial to cell fitness, evolution and survival in the recently discovered error-prone DNA polymerases. Large numbers of errant DNA polymerases, spanning microorganisms to humans, are used to rescue stalled replication forks by copying damaged DNA and even undamaged DNA to generate 'purposeful' mutations that generate genetic diversity in times of stress. Here we focus on low-fidelity polymerases from bacteria, comparing Escherichia coli, archeabacteria and those most recently discovered in Gram-positive Bacilli, Streptococcus, pathogenic Mycobacterium and intein-containing cyanobacteria.

Adaptation, Biological↗
PubMed2003

Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair.

Protein clamps are ubiquitous and essential components of DNA metabolic machineries, where they serve as mobile platforms that interact with a large variety of proteins. In this report we identify residues that are required for binding of the beta-clamp to DNA polymerase III of Escherichia coli, a polymerase of the Pol C family. We show that the alpha polymerase subunit of DNA polymerase III interacts with the beta-clamp via its extreme seven C-terminal residues, some of which are conserved. Moreover, interaction of Pol III with the clamp takes place at the same site as that of the delta-subunit of the clamp loader, providing the basis for a switch between the clamp loading machinery and the polymerase itself. Escherichia coli DNA polymerases I, II, IV and V (UmuC) interact with beta at the same site. Given the limited amounts of clamps in the cell, these results suggest that clamp binding may be competitive and regulated, and that the different polymerases may use the same clamp sequentially during replication and repair.

Amino Acid Sequence↗
PubMed2003

Escherichia coli DNA polymerase V subunit exchange: a post-SOS mechanism to curtail error-prone DNA synthesis.

DNA polymerase V consisting of a heterotrimer composed of one molecule of UmuC and two molecules of UmuD' (UmuD'2C) is responsible for SOS damage-induced mutagenesis in Escherichia coli. Here we show that although the UmuD'2C complex remains intact through multiple chromatographic steps, excess UmuD, the precursor to UmuD', displaces UmuD' from UmuD'2C by forming a UmuDD' heterodimer, while UmuC concomitantly aggregates as an insoluble precipitate. Although soluble UmuD'2C is readily detected when the two genes are co-transcribed and translated in vitro, soluble UmuD2C or UmuDD'C are not detected. The subunit exchange between UmuD'2C and UmuD offers a biological means to inactivate error-prone polymerase V following translesion synthesis, thus preventing mutations from occurring on undamaged DNA.

Cross-Linking Reagents↗
PubMed2003

The essential C family DnaE polymerase is error-prone and efficient at lesion bypass.

DnaE-type DNA polymerases belong to the C family of DNA polymerases and are responsible for chromosomal replication in prokaryotes. Like most closely related Gram-positive cells, Streptococcus pyogenes has two DnaE homologs Pol C and DnaE; both are essential to cell viability. Pol C is an established replicative polymerase, and DnaE has been proposed to serve a replicative role. In this report, we characterize S. pyogenes DnaE polymerase and find that it is highly error-prone. DnaE can bypass coding and noncoding lesions with high efficiency. Error-prone extension is accomplished by either of two pathways, template-primer misalignment or direct primer extension. The bypass of abasic sites is accomplished mainly through "dNTP-stabilized" misalignment of template, thereby generating (-1) deletions in the newly synthesized strand. This mechanism may be similar to the dNTP-stabilized misalignment mechanism used by the Y family of DNA polymerases and is the first example of lesion bypass and error-prone synthesis catalyzed by a C family polymerase. Thus, DnaE may function in an error-prone capacity that may be essential in Gram-positive cells but not Gram-negative cells, suggesting a fundamental difference in DNA metabolism between these two classes of bacteria.

2-Aminopurine↗
PubMed2003

Computer simulation of the chemical catalysis of DNA polymerases: discriminating between alternative nucleotide insertion mechanisms for T7 DNA polymerase.

Understanding the chemical step in the catalytic reaction of DNA polymerases is essential for elucidating the molecular basis of the fidelity of DNA replication. The present work evaluates the free energy surface for the nucleotide transfer reaction of T7 polymerase by free energy perturbation/empirical valence bond (FEP/EVB) calculations. A key aspect of the enzyme simulation is a comparison of enzymatic free energy profiles with the corresponding reference reactions in water using the same computational methodology, thereby enabling a quantitative estimate for the free energy of the nucleotide insertion reaction. The reaction is driven by the FEP/EVB methodology between valence bond structures representing the reactant, pentacovalent intermediate, and the product states. This pathway corresponds to three microscopic chemical steps, deprotonation of the attacking group, a nucleophilic attack on the P(alpha) atom of the dNTP substrate, and departure of the leaving group. Three different mechanisms for the first microscopic step, the generation of the RO(-) nucleophile from the 3'-OH hydroxyl of the primer, are examined: (i) proton transfer to the bulk solvent, (ii) proton transfer to one of the ionic oxygens of the P(alpha) phosphate group, and (iii) proton transfer to the ionized Asp654 residue. The most favorable reaction mechanism in T7 pol is predicted to involve the proton transfer to Asp654. This finding sheds light on the long standing issue of the actual role of conserved aspartates. The structural preorganization that helps to catalyze the reaction is also considered and analyzed. The overall calculated mechanism consists of three subsequent steps with a similar activation free energy of about 12 kcal/mol. The similarity of the activation barriers of the three microscopic chemical steps indicates that the T7 polymerase may select against the incorrect dNTP substrate by raising any of these barriers. The relative height of these barriers comparing right and wrong dNTP substrates should therefore be a primary focus of future computational studies of the fidelity of DNA polymerases.

Aspartic Acid↗
PubMed2003

Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation.

Activation-induced cytidine deaminase (AID) is a protein required for B cells to undergo class switch recombination and somatic hypermutation (SHM)--two processes essential for producing high-affinity antibodies. Purified AID catalyses the deamination of C to U on single-stranded (ss)DNA. Here, we show in vitro that AID-catalysed C deaminations occur preferentially on 5' WRC sequences in accord with SHM spectra observed in vivo. Although about 98% of DNA clones suffer no mutations, most of the remaining mutated clones have 10-70 C to T transitions per clone. Therefore, AID carries out multiple C deaminations on individual DNA strands, rather than jumping from one strand to another. The avid binding of AID to ssDNA could result from its large net positive charge (+11) at pH 7.0, owing to a basic amino-terminal domain enriched in arginine and lysine. Furthermore, AID exhibits a 15-fold preference for C deamination on the non-transcribed DNA strand exposed by RNA polymerase than the transcribed strand protected as a RNA-DNA hybrid. These deamination results on ssDNA bear relevance to three characteristic features of SHM: preferential mutation at C sites within WRC hotspot sequences, the broad clonal mutagenic heterogeneity of antibody variable regions targeted for mutation, and the requirement for active transcription to obtain mutagenesis.

Amination↗
PubMed2003

Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase.

The expression of activation-induced cytidine deaminase (AID) is prerequisite to a "trifecta" of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA-DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC --> dU deamination activity most avidly on double-stranded DNA substrates containing a small "transcription-like" single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.

Animals↗
PubMed2003

Mechanism of loading the Escherichia coli DNA polymerase III beta sliding clamp on DNA. Bona fide primer/templates preferentially trigger the gamma complex to hydrolyze ATP and load the clamp.

The Escherichia coli DNA polymerase III gamma complex clamp loader assembles the ring-shaped beta sliding clamp onto DNA. The core polymerase is tethered to the template by beta, enabling processive replication of the genome. Here we investigate the DNA substrate specificity of the clamp-loading reaction by measuring the pre-steady-state kinetics of DNA binding and ATP hydrolysis using elongation-proficient and deficient primer/template DNA. The ATP-bound clamp loader binds both elongation-proficient and deficient DNA substrates either in the presence or absence of beta. However, elongation-proficient DNA preferentially triggers gamma complex to release beta onto DNA with concomitant hydrolysis of ATP. Binding to elongation-proficient DNA converts the gamma complex from a high affinity ATP-bound state to an ADP-bound state having a 10(5)-fold lower affinity for DNA. Steady-state binding assays are misleading, suggesting that gamma complex binds much more avidly to non-extendable primer/template DNA because recycling to the high affinity binding state is rate-limiting. Pre-steady-state rotational anisotropy data reveal a dynamic association-dissociation of gamma complex with extendable primer/templates leading to the diametrically opposite conclusion. The strongly favored dynamic recognition of extendable DNA does not require the presence of beta. Thus, the gamma complex uses ATP binding and hydrolysis as a mechanism for modulating its interaction with DNA in which the ATP-bound form binds with high affinity to DNA but elongation-proficient DNA substrates preferentially trigger hydrolysis of ATP and conversion to a low affinity state.

Adenosine Triphosphate↗
PubMed2003

Computer simulation studies of the fidelity of DNA polymerases.

Computer simulations can provide in principle quantitative correlation between the structures of DNA polymerases and the replication fidelity. This paper describes our progress in this direction. Using several theoretical approaches, including the free energy perturbation (FEP), linear response approximation (LRA), and the empirical valence bond (EVB) methods, we examined the stability of several mismatched base pairs in DNA duplex in aqueous solution, the contribution of binding energy to the fidelity of DNA polymerases beta and T7, and the mechanism and energetics of the polymerization reaction catalyzed by T7 DNA polymerase.

Computer Simulation↗