PubMed Health⌕ Search

Biomedical subjects

Susan S Wallace

Publications and source records attributed to Susan S Wallace.

At least 19 recordsLinked to original sources
PubMed2007

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol.

Thymine glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-A crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5' template guanine. The position of the displaced 5' template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.

Crystallography, X-Ray↗
PubMed2006

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family.

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.

Bacteriophage T4↗
PubMed2006

Kinetics of error generation in homologous B-family DNA polymerases.

The kinetics of forming a proper Watson-Crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the B Family replicative DNA polymerase from bacteriophage T4. Structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage RB69. In this work, the homologous enzymes from RB69 and T4 were compared in parallel reactions to determine the relative abilities of the two polymerases to incorporate correct nucleotides as well as to form improper pairings. The kinetic rates for three different exonuclease mutants for each enzyme were measured for incorporation of an A opposite T and an A opposite furan as well as for the formation of A:C and T:T mismatches. The T4 exonuclease mutants were all approximately 2- to 7-fold more efficient than the corresponding RB69 exonuclease mutants depending on whether a T or furan was in the templating position and which exonuclease mutant was used. The rates for mismatch formation by T4 were significantly reduced compared with incorporation opposite furan, much more so than the corresponding RB69 mutant. These results show that there are kinetic differences between the two enzymes but they are not large enough to preclude structural assumptions for T4 DNA polymerase based on the known structure of the RB69 DNA polymerase.

Base Pair Mismatch↗
PubMed2006

Oxidative DNA glycosylases: recipes from cloning to characterization.

As new organisms are being sequenced on a daily basis, new DNA glycosylases that recognize DNA damage can be easily identified in an effort to understand both their phylogenetics and substrate specificities. As a practical matter, existing bacterial and human homologs need to be readily available as laboratory reagents in order to compare the activities of the novel enzymes to existing enzymes. This chapter attempts to provide a primer for cloning, expression, and assay procedures for bacterial and human DNA glycosylases that recognize oxidative DNA damages. These methodologies can be translated readily to novel DNA glycosylases or to DNA glycosylases that recognize other types of DNA damages.

Cloning, Molecular↗
PubMed2005

A new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress. Application to dystrophin-deficient DMD skeletal muscles.

This is the first report on the development of an immunohistochemical technique, combined with quantitative image analysis, for the assessment of oxidative stress quantitatively in nuclear DNA in situ, and its application to measure DNA damage in Duchenne muscular dystrophic (DMD) muscles. Three sequential staining procedures for cell nuclei, a cell marker, and a product of oxidative DNA damage, 8-oxoguanine (8-oxoG), were performed. First, the nuclei in muscle sections were stained with Neutral Red followed by the capture of their images with an image analysis system used for absorbance measurements. Second, the same sections were then immunostained for laminin in basement membranes as the cell marker. Next, the sections were treated with 2 N HCl to remove the bound Neutral Red and to denature tissue DNA. Third, the sections were immunostained for 8-oxoG in DNA, using diaminobenzidine (DAB) to reveal the antibody complex. This was followed by capture of the images of the immunostained sections as previously. The absorbances at 451.2 nm of bound Neutral Red and DAB polymer oxides, the final product of 8-oxoG immunostaining, were measured in the same myonuclei in the sections. Analysis of these absorbances permitted indices of the 8-oxoG content, independent of the nuclear densities, to be determined in nuclear DNA in single myofibres and myosatellite cells surrounded by basement membranes. We found that the mean index for the myonuclei in biceps brachii muscles of 2- to 7-year-old patients was 14% higher than that in age-matched normal controls. This finding of the increased oxidative stress in the myonuclei in young DMD muscles agrees with the previous reports of increased oxidative stress in the cytoplasm in the DMD myofibres and myosatellite cells. The present technique for the quantitative assessment of oxidative stress in nuclear DNA in situ is applicable not only in biomedical research but also in the development of effective drugs for degenerative diseases related to oxidative stress.

Animals↗
PubMed2005

Base excision repair by hNTH1 and hOGG1: a two edged sword in the processing of DNA damage in gamma-irradiated human cells.

Using siRNA technology, we down-regulated in human B-lymphoblastoid TK6 cells the two major oxidative DNA glycosylases/AP lyases that repair free radical-induced base damages, hNTH1 and hOGG1. The down-regulation of hOGG1, the DNA glycosylase whose main substrate is the mutagenic but not cytotoxic 8-oxoguanine, resulted in reduced radiation cytotoxicity and decreased double strand break (DSB) formation post-irradiation. This supports the idea that the oxidative DNA glycosylases/AP lyases convert radiation-induced clustered DNA lesions into lethal DSBs and is in agreement with our previous finding that overexpression of hNTH1 and hOGG1 in TK6 cells increased radiation lethality, mutant frequency at the thymidine kinase locus and the enzymatic production of DSBs post-irradiation [N. Yang, H. Galick, S.S. Wallace, Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair (Amst) 3 (2004) 1323-1334]. Interestingly, cells deficient in hNTH1, the DNA glycosylase that repairs a major lethal single free radical damage, thymine glycol, were more radiosensitive but at the same time fewer DSBs were formed post-irradiation. These results indicate that hNTH1 plays two roles in the processing of radiation damages: repair of potentially lethal single lesions and generation of lethal DSBs at clustered damage sites. In contrast, in hydrogen peroxide-treated cells where the majority of free radical DNA damages are single lesions, the base excision repair pathway functioned to protect the cells. Here, overexpression of hNTH1 and hOGG1 resulted in reduced cell killing while suppression of glycosylase expression resulted in elevated cell death.

B-Lymphocytes↗
PubMed2005

Engineering functional changes in Escherichia coli endonuclease III based on phylogenetic and structural analyses.

Escherichia coli endonuclease III (EcoNth) plays an important cellular role by removing premutagenic pyrimidine damages produced by reactive oxygen species. EcoNth is a bifunctional enzyme that has DNA glycosylase and apurinic/apyrimidinic lyase activities. Using a phylogeny of natural sequences, we selected to study EcoNth serine 39, aspartate 44, and arginine 184, which are presumed to be in the vicinity of the damaged base in the glycosylase-substrate complex. These three amino acids are highly conserved among Nth orthologs, although not among homologous glycosylases, such as MutY, that have different base specificities and no lyase activity. To examine the role of these amino acids in catalysis, we constructed three mutants of EcoNth, in which Ser39 was replaced with leucine (S39L), Asp44 was replaced with valine (D44V), and Arg184 was replaced with alanine (R184A), which are the corresponding residues in EcoMutY. We showed that EcoNth S39L does not have significant glycosylase activity for oxidized pyrimidines, although it maintained AP lyase activity. In contrast, EcoNth D44V retained glycosylase activity against oxidized pyrimidines, but the apparent rate constant for the lyase activity of EcoNth D44V was significantly lower than that of EcoNth, indicating that Asp44 in EcoNth is required for beta-elimination. Finally, EcoNth R184A maintained lyase activity but exhibited glycosylase specificity different from that of EcoNth. The functional consequences of each of these three substitutions can be rationalized in the context of high resolution protein structures. Thus phylogeny-based scanning mutagenesis has allowed us to identify novel roles for amino acids in the substrate binding pocket of EcoNth in base recognition and/or catalysis.

Arginine↗
PubMed2005

Solution-state NMR investigation of DNA binding interactions in Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg): a dynamic description of the DNA/protein interface.

Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair (BER) protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free in solution and bound to a double-stranded DNA oligomer containing 1,3-propanediol (13-PD), a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 251 (83%) free-precession 15N/1H HSQC cross peaks were observed and 180 of these were assignable, indicating that approximately 30% of the residues undergo intermediate motion on the NMR timescale, broadening the resonances beyond detection or making them intractable in backbone assignment experiments. The majority of these affected residues were in the polypeptide linker region and the interface between the N- and C-terminal domains. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate timescale motion observed for free Fpg, including those residues directly involved in DNA binding, notwithstanding a nanomolar dissociation constant for 13-PD binding. Indeed, after binding to 13-PD, at least approximately 40% of the Fpg residues undergo intermediate timescale motion even though all other residues exhibit tight DNA binding characteristic of slow exchange. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that were quenched upon binding 13-PD. In free Fpg, heteronuclear 1H-15N NOE experiments detected picosecond timescale backbone motion in the alphaF-beta9 loop, the region primarily responsible for chemically discriminating 8-oxoguanine (8-oxoG) over normal guanine, that was quenched after binding 13-PD. Collectively, these observations reveal that, in solution, Fpg is a very dynamic molecule even after binding damaged DNA. Such motion, especially at the DNA binding surface, may be key to its processive search for DNA damage and its catalytic functions once it recognizes damaged DNA.

Amino Acid Sequence↗
PubMed2005

Bumps in the road: how replicative DNA polymerases see DNA damage.

Significant advances have been made recently in the study of polymerases. First came the realization that there are many more DNA polymerases than originally thought; indeed, no fewer than 14 template-dependent DNA polymerases are found in mammals. Concurrent structural studies of DNA polymerases bound to DNA and incoming nucleotide have revealed how these remarkable copying machines select the correct deoxynucleoside triphosphate among a sea of nucleotides. A whole new level of insight into DNA replication fidelity has been reached as a result of recently determined crystal structures of DNA lesions in the context of the active sites of repair, replicative and specialized DNA polymerases. These structures illustrate why some lesions can be bypassed readily, whereas others are strong blocks to DNA replication.

Binding Sites↗
PubMed2004

Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks.

A significant proportion of cellular DNA damages induced by ionizing radiation are produced in clusters, also called multiply damaged sites. It has been demonstrated by in vitro studies and in bacteria that clustered damage sites can be converted to lethal double strand breaks by oxidative DNA glycosylases during attempted base excision repair. To determine whether DNA glycosylases could produce double strand breaks at radiation-induced clustered damages in human cells, stably transformed human lymphoblastoid TK6 cells that inducibly overexpress the oxidative DNA glycosylases/AP lyases, hNTH1 and hOGG1, were assessed for their radiation responses, including survival, mutation induction and the enzymatic production of double strand breaks post-irradiation. We found that additional double strand breaks were generated during post-irradiation incubation in uninduced TK6 control cells. Moreover, overproduction of either DNA glycosylase resulted in significantly increased double strand break formation, which correlated with an elevated sensitivity to the cytotoxic and mutagenic effects of ionizing radiation. These data show that attempted repair of radiation damage, presumably at clustered damage sites, by the oxidative DNA glycosylases can lead to the formation of potentially lethal and mutagenic double strand breaks in human cells.

B-Lymphocytes↗
PubMed2004

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity.

In prokaryotes, two DNA glycosylases recognize and excise oxidized pyrimidines: endonuclease III (Nth) and endonuclease VIII (Nei). The oxidized purine 8-oxoguanine, on the other hand, is recognized by Fpg (also known as MutM), a glycosylase that belongs to the same family as Nei. The recent availability of the human genome sequence allowed the identification of three human homologs of Escherichia coli Nei. We report here the crystal structure of a human Nei-like (NEIL) enzyme, NEIL1. The structure of NEIL1 exhibits the same overall fold as E. coli Nei, albeit with an unexpected twist. Sequence alignments had predicted that NEIL1 would lack a zinc finger, and it was therefore expected to use a different DNA-binding motif instead. Our structure revealed that, to the contrary, NEIL1 contains a structural motif composed of two antiparallel beta-strands that mimics the antiparallel beta-hairpin zinc finger found in other Fpg/Nei family members but lacks the loops that harbor the zinc-binding residues and, therefore, does not coordinate zinc. This "zincless finger" appears to be required for NEIL1 activity, because mutating a very highly conserved arginine within this motif greatly reduces the glycosylase activity of the enzyme.

Crystallography↗
PubMed2004

Overproduction, crystallization and preliminary crystallographic analysis of a novel human DNA-repair enzyme that recognizes oxidative DNA damage.

DNA glycosylases repair oxidative DNA damage caused by free radicals. Recently, NEIL1, a human homolog of Escherichia coli DNA glycosylase endonuclease VIII, has been identified and shown to exhibit broad substrate specificity for a variety of types of pyrimidine-base damage. An active C-terminal deletion construct of NEIL1 was overexpressed in E. coli and crystallized. The unliganded NEIL1 crystallizes in space group R3, with unit-cell parameters a = b = 132.2, c = 51.1 A. Complete data sets were collected from native, selenomethionyl and iodinated NEIL1 to 2.1, 2.3 and 2.4 angstroms, respectively.

Crystallography, X-Ray↗
PubMed2004

Crystallographic snapshots of a replicative DNA polymerase encountering an abasic site.

Abasic sites are common DNA lesions, which are strong blocks to replicative polymerases and are potentially mutagenic when bypassed. We report here the 2.8 A structure of the bacteriophage RB69 replicative DNA polymerase attempting to process an abasic site analog. Four different complexes were captured in the crystal asymmetric unit: two have DNA in the polymerase active site whereas the other two molecules are in the exonuclease mode. When compared to complexes with undamaged DNA, the DNA surrounding the abasic site reveals distinct changes suggesting why the lesion is so poorly bypassed: the DNA in the polymerase active site has not translocated and is therefore stalled, precluding extension. All four molecules exhibit conformations that differ from the previously published structures. The polymerase incorporates dAMP across the lesion under crystallization conditions, indicating that the different conformations observed in the crystal may be part of the active site switching reaction pathway.

Binding Sites↗
PubMed2004

Single-stranded breaks in DNA but not oxidative DNA base damages block transcriptional elongation by RNA polymerase II in HeLa cell nuclear extracts.

Transcription and repair of many DNA helix-distorting lesions such as cyclobutane pyrimidine dimers have been shown to be coupled in cells across phyla from bacteria to humans. The signal for transcription-coupled repair appears to be a stalled transcription complex at the lesion site. To determine whether oxidative DNA lesions can block correctly initiated human RNA polymerase II, we examined the effect of site-specifically introduced oxidative damages on transcription in HeLa cell nuclear extracts. We found that transcription was blocked by single-stranded breaks, common oxidative DNA lesions, when present in the transcribed strand of the transcription template. Cyclobutane pyrimidine dimers, which have been previously shown to block transcription both in vitro and in vivo, also blocked transcription in the HeLa cell nuclear transcription assay. In contrast, the oxidative DNA base lesions, 8-oxoguanine, 5-hydroxycytosine, and thymine glycol did not inhibit transcription, although pausing was observed with the thymine glycol lesion. Thus, DNA strand breaks but not oxidative DNA base damages blocked transcription by RNA polymerase II.

Cell Extracts↗
PubMed2003

Decline of nuclear and mitochondrial oxidative base excision repair activity in late passage human diploid fibroblasts.

There are numerous studies documenting the increase of oxidative DNA damage in the nuclei and mitochondria of senescing cells as well as in tissues of aging animals. Here, we show that in IMR 90 human diploid fibroblasts, DNA repair activity is robust in both nuclear and mitochondrial extracts, however, the levels of activity differed against the three substrates tested. In extracts, cleavage of the 8-oxoguanine substrate, and to a lesser extent the dihydrouracil-containing substrate, occurred in a concerted reaction between the DNA glycosylases and the second enzyme in the reaction, hAPE. Cleavage of both the furan and the dihydrouracil-containing substrates was unchanged when nuclear extracts from early and late passage cells were compared. However, cleavage of the 8-oxoguanine substrate was substantially reduced in the nuclear extracts from late passage cells and significantly reduced transcription from the hOGG1 gene was observed. When mitochondrial extracts were examined, activity on all three substrates was significantly reduced, with the reduction in hAPE activity being the most marked. The reduction in cleavage of the furan substrate was not simply due to inactive mitochondrial AP endonuclease but a substantially reduced amount of hAPE protein; transcription from the hAPE gene was also reduced. Confocal microscopic analysis confirmed that hAPE was present in the mitochondria of early passage cells but greatly reduced in the mitochondria of late passage cells. Cytoplasmic extracts from late passage fibroblasts also showed reduced activity with all three substrates suggesting that the residual hAPE, and activities that recognized dihydrouracil, were preferentially targeted to the nuclei. Taken together the data support the concept that the increase in oxidative damage in the mitochondrial DNA of senescing cells and tissues from aging animals is due to reduced base excision repair activity.

Cell Nucleus↗
PubMed2002

Human DNA polymerase kappa bypasses and extends beyond thymine glycols during translesion synthesis in vitro, preferentially incorporating correct nucleotides.

Human polymerase kappa (polkappa), the product of the human POLK (DINB1) gene, is a member of the Y superfamily of DNA polymerases that support replicative bypass of chemically modified DNA bases (Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo, T., Walker, G. C., Wang, Z., and Woodgate, R. (2001) Mol. Cell 8, 7-8; Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V., and Friedberg, E. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11922-11927). Polkappa is shown here to bypass 5,6-dihydro-5,6-dihydroxythymine (thymine glycol) generated in two different DNA substrate preparations. Polkappa inserts the correct base adenine opposite thymine glycol in preference to the other three bases. Additionally, the enzyme correctly extends beyond the site of the thymine glycol lesion when presented with adenine opposite thymine glycol at the primer terminus. However, steady state kinetic analysis of nucleotides incorporated opposite thymine glycol demonstrates different misincorporation rates for guanine with each of the two DNA substrates. The two substrates differ only in the relative proportions of thymine glycol stereoisomers, suggesting that polkappa distinguishes among stereoisomers and exhibits reduced discrimination between purines when incorporating a base opposite a 5R thymine glycol stereoisomer. When extending beyond the site of the lesion, the misincorporation rate of polkappa for each of the three incorrect nucleotides (adenine, guanine, and thymine) is dramatically increased. Our findings suggest a role for polkappa in both nonmutagenic and mutagenic bypass of oxidative damage.

Adenine↗
PubMed2002

A novel human DNA glycosylase that removes oxidative DNA damage and is homologous to Escherichia coli endonuclease VIII.

Prokaryotes and lower eukaryotes possess redundant activities that remove the plethora of oxidative DNA base damages produced during normal oxidative metabolism and which have been associated with cancer and aging. Thus far, only one oxidized pyrimidine-specific DNA glycosylase has been identified in humans, hNthl. Here, we report the identification of three new putative human DNA glycosylases that are phylogenetically members of the Fpg/Nei family primarily found in the bacterial kingdom. We have characterized one of these, hNEI1, and show it to be functionally homologous to bacterial Nei, that is, its principal substrates are oxidized pyrimidines, it undergoes a lyase reaction by, beta,delta-elimination and traps a Schiff base with a substrate containing thymine glycol (Tg). Furthermore, inactivation of active site residues shown to be important in Escherichia coli Nei inactivate the human enzyme. The hNEI1 gene is located on the long arm of chromosome 15 that is frequently deleted in human cancers.

Amino Acid Sequence↗