MutM, a protein that prevents G.C----T.A transversions, is formamidopyrimidine-DNA glycosylase.

We have cloned chromosomal DNA bordering an insert that inactivates mutM. Sequencing of this clone has revealed that the insertion element is located between the promoter and structural gene for formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase). An overproducing clone of Fapy-DNA glycosylase complements the original mutM strain that had been isolated after EMS mutagenesis. Thus, we conclude that MutM is actually Fapy-DNA glycosylase. mutM has previously been characterized as a mutator strain that leads specifically to G.C----T.A transversions. This in vivo characterization correlates well with the mutagenic potential of one of the lesions Fapy-DNA glycosylase removes, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-OxodG)

The Fpg/Nei Family of DNA Glycosylases

During the initial stages of the base excision DNA repair (BER) pathway, DNA glycosylases are responsible for locating and removing the majority of endogenous oxidative base lesions. The bifunctional formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of the Fpg/Nei family, one of the two families of glycosylases that recognize oxidized DNA bases, the other being the HhH/GPD (or Nth) superfamily. Structural and biochemical developments over the past decades have led to novel insights into the mechanism of damage recognition by the Fpg/Nei family of enzymes. Despite the overall structural similarity among members of this family, these enzymes exhibit distinct features that make them unique. This review summarizes the current structural knowledge of the Fpg/Nei family members, emphasizes their substrate specificities, and describes how these enzymes search for lesions

Strukturní studie opravy abazického místa a tvorba kovalentního spojení opačných řetězců DNA

DNA damage refers to any alteration or modification in the DNA structure that deviates from its natural state. Abasic site (Ap site) is one of the most common DNA lesions resulting from spontaneous depurination/depyrimidination or enzymatic base excision. When left unrepaired it can lead to a cascade of genetic mutations, potentially causing diseases like cancer. Understanding DNA repair mechanisms is vital for medical research and applications. Bacterial MutM is a DNA repair glycosylase, removing DNA damage generated by oxidative stress and preventing mutations and genomic instability. MutM belongs to the Fpg/Nei family of procaryotic enzymes, sharing structural and functional similarities with their eukaryotic counterparts, such as NEIL1-NEIL3. Here, I present two crystal structures of MutM from pathogenic Neisseria meningitidis: MutM holoenzyme and MutM bound to DNA. The free enzyme exists in an open conformation, while upon binding to DNA, both the enzyme and DNA undergo substantial structural changes and domain rearrangement. One of the DNA lesion repaired by MutM is the Ap site, which, if not repaired, may spontaneously lead to the formation of an abasic site interstrand crosslink (Ap-ICL) with an adjacent adenine in the opposite strand. NEIL3 glycosylase is known to remove Ap-ICL. With a...Poškozením DNA se rozumí jakákoli změna nebo modifikace struktury DNA, která se odchyluje od jejího přirozeného stavu. Abasické místo (Ap místo) je jedním z nejčastějších poškození DNA, které vzniká spontánní depurinací/depyrimidinací nebo enzymatickým odstraněním báze. Pokud se neopraví, může vést ke genetické mutaci a potenciálně způsobit onemocnění, jako je například rakovina. Pochopení mechanismu opravy DNA je zásadní pro lékařský výzkum a aplikaci. Bakteriální MutM je glykosyláza opravující DNA, která odstraňuje poškození DNA vzniklé oxidačním stresem a zabraňuje mutacím a genomové nestabilitě. MutM patří do rodiny prokaryotických enzymů Fpg/Nei a je strukturně i funkčně podobná se svým eukaryotickým protějškům, jako jsou NEIL1-NEIL3. Zde prezentuji dvě krystalové struktury MutM z patogenní Neisseria meningitidis: MutM holoenzym a MutM vázaný na DNA. Volný enzym existuje v otevřené konformaci, zatímco po vazbě na DNA, dochází k podstatným strukturním změnám a přeskupení domén enzymu i ohybu DNA. Jednou z poškození DNA opravovaných MutM je Ap místo, které, pokud není opraveno, může spontánně vést k vytvoření mezivláknového kovanetního prokřízení DNA (Ap-ICL) se sousedním adeninem na opačném vlákně DNA. Je známo, že glykosyláza NEIL3 odstraňuje Ap-ICL. Pomocí série různých oligonukleotidů jsme...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemieFaculty of SciencePřírodovědecká fakult

Strukturní studie opravy abazického místa a tvorba kovalentního spojení opačných řetězců DNA

DNA damage refers to any alteration or modification in the DNA structure that deviates from its natural state. Abasic site (Ap site) is one of the most common DNA lesions resulting from spontaneous depurination/depyrimidination or enzymatic base excision. When left unrepaired it can lead to a cascade of genetic mutations, potentially causing diseases like cancer. Understanding DNA repair mechanisms is vital for medical research and applications. Bacterial MutM is a DNA repair glycosylase, removing DNA damage generated by oxidative stress and preventing mutations and genomic instability. MutM belongs to the Fpg/Nei family of procaryotic enzymes, sharing structural and functional similarities with their eukaryotic counterparts, such as NEIL1-NEIL3. Here, I present two crystal structures of MutM from pathogenic Neisseria meningitidis: MutM holoenzyme and MutM bound to DNA. The free enzyme exists in an open conformation, while upon binding to DNA, both the enzyme and DNA undergo substantial structural changes and domain rearrangement. One of the DNA lesion repaired by MutM is the Ap site, which, if not repaired, may spontaneously lead to the formation of an abasic site interstrand crosslink (Ap-ICL) with an adjacent adenine in the opposite strand. NEIL3 glycosylase is known to remove Ap-ICL. With a...Poškozením DNA se rozumí jakákoli změna nebo modifikace struktury DNA, která se odchyluje od jejího přirozeného stavu. Abasické místo (Ap místo) je jedním z nejčastějších poškození DNA, které vzniká spontánní depurinací/depyrimidinací nebo enzymatickým odstraněním báze. Pokud se neopraví, může vést ke genetické mutaci a potenciálně způsobit onemocnění, jako je například rakovina. Pochopení mechanismu opravy DNA je zásadní pro lékařský výzkum a aplikaci. Bakteriální MutM je glykosyláza opravující DNA, která odstraňuje poškození DNA vzniklé oxidačním stresem a zabraňuje mutacím a genomové nestabilitě. MutM patří do rodiny prokaryotických enzymů Fpg/Nei a je strukturně i funkčně podobná se svým eukaryotickým protějškům, jako jsou NEIL1-NEIL3. Zde prezentuji dvě krystalové struktury MutM z patogenní Neisseria meningitidis: MutM holoenzym a MutM vázaný na DNA. Volný enzym existuje v otevřené konformaci, zatímco po vazbě na DNA, dochází k podstatným strukturním změnám a přeskupení domén enzymu i ohybu DNA. Jednou z poškození DNA opravovaných MutM je Ap místo, které, pokud není opraveno, může spontánně vést k vytvoření mezivláknového kovanetního prokřízení DNA (Ap-ICL) se sousedním adeninem na opačném vlákně DNA. Je známo, že glykosyláza NEIL3 odstraňuje Ap-ICL. Pomocí série různých oligonukleotidů jsme...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemieFaculty of SciencePřírodovědecká fakult

Biochemical Characterization of DNA Glycosylases from Mycobacterium Tuberculosis

The DNA glycosylases function in the first step of the base excision repair (BER) process, that is responsible for removing base lesions resulting from oxidation, alkylation or deamination. The DNA glycosylases that recognize oxidative base damage fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis. While Fpg proteins are widely distributed among the bacteria and plants, Nei homologs are sparsely distributed across phyla, and are only found in γ-proteobacteria, actinobacteria and metazoans. Interestingly, M. tuberculosis H37Rv harbors two proteins (Rv2464c and Rv3297) from the Nei clade and two (Rv2924c and Rv0944) from the Fpg clade. All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 (Rv2924c) has a substrate specificity similar to that of EcoFpg and recognizes oxidized purines. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpg1 has a substantially increased opposite base discrimination compared to EcoFpg. The Rv0944 gene encodes MtuFpg2, which contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5- OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (5S) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Escherichia coli, the protein decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M. tuberculosis against oxidative stress, as has been observed with other pathogens

The construction and phenotypic characterization of mycobacterial mutants deficient in DNA glycosylases

Mycobacterium tuberculosis is an exquisitely adapted intracellular pathogen that encounters hostile, host-derived reactive nitrogen and oxygen intermediates during the course of infection of its human host. These radicals cause DNA damage, which is repaired through various pathways to allow for the continued survival of the organism. Base excision repair (BER) is one such pathway, which depends on DNA glycosylases to identify and excise damaged DNA bases. Formamidopyrimidine DNA glycosylase (Fpg/ MutM/ FAPY) and Endonuclease VIII (Nei) are such enzymes, which both target oxidatively damaged DNA and together, form the Fpg family of DNA glycosylases. Bioinformatic analyses identified two copies each of Fpg and Nei-encoding genes in M. tuberculosis as well as in its non-pathogenic relative, Mycobacterium smegmatis. To understand the role of these multiple glycosylases in the maintenance of genomic integrity and survival of mycobacteria, the genes encoding the four Fpg/Nei glycosylases were individually deleted in M. smegmatis strain mc2155 by homologous recombination. In addition to the four single mutants, double and triple Fpg and Nei glycosylase knockout mutants were generated by sequential gene knockout. When compared to the parental strain, the single and double mutants showed no variation in growth kinetics, no increased sensitivity to hydrogen peroxide and no increase in spontaneous mutation rates. However, a slight increase in frequency of spontaneous C T transition mutations was observed in double knockout mutants compared to the wild type and single mutant strains. These results suggest that these enzymes may be part of an extensive network of enzymes which collectively work to enhance the overall survival of M. smegmatis through the repair of oxidatively damaged DNA

Functional Exploration and Characterization of the Deaminases of Cog0402

High throughput sequencing technology and availability of this information has changed the way enzyme families can be studied. Sequence information from large public databases such as GenBank and UniProtKB can easily retrieved for the purpose of identifying unique enzymatic activities. The strategy adopted for this study is to identify characterized enzymes and the sequence features which give rise to their substrate specificity. Homologues of these enzymes are retrieved, and any active site variations can be readily identified. Cluster of Orthologous Groups (cog) 0402 is a family of enzymes which comprise a portion of the amidohydrolase superfamily. This group catalyzes a deamination reaction, releasing free ammonia and replacing it with a tautomerized oxygen. Cog0402 is most well known for guanine and cytosine deaminase, however other functions exist. One such function was that of S-adenosylhomocysteine deaminase, which was related to a large group of uncharacterized enzymes. These enzymes were predicted by us to deaminate 5’-modified adenosines. The enzymes were physically characterized these predictions were confirmed and a 5’-deoxyadenosine deaminase was discovered in addition to an 8-oxoadenine deaminase. During this study it was noted that background isoguanine deaminase activity was found at appreciable rates in E. coli. This activity was purified and identified using nanoLC-MS/MS and found to be caused by E. coli cytosine deaminase. E. coli cytosine deaminase itself is found in a cluster of uncharacterized enzymes with a single amino acid difference in the active site. Representative enzymes were purified and a 5-methylcytosine deaminase was discovered. This enzyme is capable of rescuing thymine auxotrophs in the presence of 5-methylcytosine, and will confer sensitivity to 5-fluorocytosine. Finally, an enzyme distantly related to cytosine deaminase was purified and found to be a unique pterin deaminase. It was most efficient for oxidized pterin rings and would accept a variety of substituents on the C6 positions. Futhermore, it was thought to catalyze the first step of an undescribed pterin degradation pathway

Human DNA repair enzyme O6-Methylguanine-DNA Methyltransferase in cellular regulation upon DNA damage

Ph.DDOCTOR OF PHILOSOPH

The role of base excision repair proteins in the cellular responses to the anticancer drug cisplatin

Thesis (Ph.D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2000.Includes bibliographical references.by Maria Kartalou.Ph.D

Base Excision Repair in Chromatin

ABSTRACT DNA in the eukaryotic nucleus is complexed with histone and non-histone proteins into chromatin. Nucleosomes, the basic repeating unit of chromatin, not only package DNA but are also intimately involved the regulation of gene expression. All DNA transactions including replication, transcription, recombination and repair take place in such a chromatin environment. Access to packaged nucleosomal DNA in vivo is mediated at least in part by protein complexes that modify or remodel chromatin. Buried sequences in nucleosomes can also transiently become accessible to DNA binding proteins during cycles of partial, reversible unwrapping of nucleosomal DNA from the histone octamer. We have investigated the ability of the human, bifunctional DNA glycosylase, endonuclease III (hNTH1), to initiate base excision repair (BER) of discretely positioned oxidative lesions in model nucleosomes. hNTH1 was able to process a thymine glycol (Tg) lesion almost as efficiently as naked DNA, when the minor groove of the lesion faced away from the histone octamer. Lesion processing did not require or result in detectable nucleosome disruption, as assayed in gel mobility-shift experiments. Instead, hNTH1 formed a slower migrating enzyme-nucleosome ternary complex that was found to contain processed DNA. Processing of an inward-facing Tg residue located just 5 bp away from the outward-facing lesion was much reduced and processing of a sterically occluded Tg residue positioned closer to the dyad center of the nucleosome was even more reduced. Notably, processing of both inward-facing lesions was found to increase as a function of enzyme concentration. Restriction enzyme protection studies indicated that access to these inward-facing lesions did not entail nucleosomal translocation or sliding. Collectively, these observations are consistent with a model in which hNTH1 binds to lesions during cycles of reversible, partial unwrapping of nucleosomal DNA from the histone octamer core. To further investigate this partial unwrapping hypothesis, we studied the kinetics of hNTH1 processing of sterically occluded lesions in greater detail. Our results suggest that efficiency of processing of inward-facing lesions is a function of both DNA unwrapping and rewrapping rates, and enzyme affinity for the lesion. In addition, we determined that APE1 which catalyzes the second step in BER, exhibited an increasing capacity to process inward-facing furan residues as its concentration was increased. Thus as with hNTH1, we hypothesize that APE1 can capture occluded furan residues during cycles of partial DNA unwrapping. We propose that cellular regulatory factors benefit from this intrinsic, periodic exposure of nucleosomal DNA exposure in vivo, which may be amplified by the downstream recruitment of remodeling and / or modifying proteins to facilitate DNA transactions in the cell