Insight into the Roles of Tyrosine 82 and Glycine 253 in the <i>Escherichia coli</i> Adenine Glycosylase MutY

Livingston, Alison L.; Kundu, Sucharita; Pozzi, Michelle Henderson; Anderson, David W.; David, Sheila S.

doi:10.1021/bi050976u

Cited by 38 publications

(58 citation statements)

References 72 publications

(165 reference statements)

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“…3) despite the fact that their catalytic residues were intact. However, prior ensemble studies with the E. coli MutY glycosylase wedge mutant proteins containing different amino acid substitutions showed a number of distinct effects on enzymatic activity as well as on binding, illustrating the multiple roles in the catalytic process undertaken by the wedge amino acid (49). As stated above, we are proposing that the mutant wedge residue still inserts into the DNA helix and may play a role in recognition of a damaged base.…”

Section: Discussionmentioning

confidence: 65%

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

Nelson

Dunn

Kathe

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

113

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DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism's DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key "wedge residue" to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it.DNA repair | search mechanisms O xidative DNA damage is produced endogenously during normal cellular metabolism or exogenously by chemical agents and ionizing radiation (1, 2). Oxidatively induced DNA damage resulting from attack by reactive oxygen species accounts for approximately one-half of all DNA base damages (3). Some oxidative base damages, such as thymine glycol, are blocks to DNA polymerases and thus potentially lethal; however, the majority of oxidative base lesions mispair with noncognate bases and are potentially mutagenic (4). Therefore, damaged bases must be repaired to maintain the cell's genomic integrity. With substantial in vivo steady-state levels of oxidative damages, alkylation damages, and apurinic/apyrimidinic (AP) sites among the nearly six billion normal bases, how DNA repair enzymes locate these damages in the sea of undamaged bases has been the subject of much speculation.The DNA repair mechanism responsible for the removal of the majority of endogenous DNA damages is the base excision repair (BER) pathway (4-6). The critical first step in BER is carried out by a DNA glycosylase that, fueled only by thermal energy, locates a damaged base and cleaves the N-glycosyl bond, thus removing the base lesion from the sugar phosphate backbone. Glycosylases are small monomeric proteins that are found in all living organisms and can be separated into different families based on substrate specificity and structural motifs. In Escherichia coli, there are three glycosylases, Nth, Fpg, and Nei, that directly remove oxidized DNA bases, and all three have an associated lyase activity that cleaves the DNA backbone. These glycosylases are members of two structural families, the helixhairpin-helix (HhH) or Nth superfamily and the helix-two turnshelix (H2TH) or Fpg/Nei family (7-9) (Fig. 1A). Interestingly, the HhH superfamily member, endonuclease III (Nth), and Fpg/ Nei family member, endonuclease VIII (Nei), primarily catalyze the removal of oxidized pyrimidines, such as 5,6-dihydroxy-5, 6-dihydrothymine (Tg), whereas formamidopyrimidine DNA glycosylase (Fpg) primarily removes oxidized purines...

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Section: Discussionmentioning

confidence: 65%

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

Nelson

Dunn

Kathe

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

113

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“…Two of the most common mutations implicated in MAP, Y165C and G382D, involve highly conserved positions in the protein. In E. coli MutY, the corresponding mutations (Y82C and G253D) lead to modest decreases in substrate binding affinity and rate of excision [77]. In addition, structural studies show that Y82 and G253 interact with the DNA near the 8-oxoguanine lesion site [39][40].…”

Section: Discussionmentioning

confidence: 99%

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

Boal

Yavin

Barton

2007

Journal of Inorganic Biochemistry

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The [4Fe-4S] cluster is ubiquitous to a class of base excision repair enzymes, in organisms ranging from bacteria to man, and was first considered as a structural element, owing to its redox stability under physiological conditions. When studied bound to DNA, two of these repair proteins (MutY and Endonuclease III from Escherichia coli) display DNA-dependent reversible electron transfer with characteristics typical of high potential iron proteins. These results have inspired a reexamination of the role of the [4Fe-4S] cluster in this class of enzymes. Might the [4Fe-4S] cluster be used as a redox cofactor to search for damaged sites using DNA-mediated charge transport, a process well known to be highly sensitive to lesions and mismatched bases? Described here are experiments demonstrating the utility of DNA-mediated charge transport in characterizing these DNA-binding metalloproteins, as well as efforts to elucidate this new function for DNA as an electronic signaling medium among the proteins.

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“…24 In the case of G:A substrates, accessing the catalytically competent intermediate must rely heavily on nonspecific electrostatic interactions to explain the sensitivity to the increased salt concentration. 36,37 In contrast, with OG:A substrates, nonspecific electrostatic interactions are not as critical for efficient adenine removal. These results underscore the importance of recognition of OG to fully engage the adenine in the active site for facile glycosidic bond cleavage.…”

mentioning

confidence: 99%

“…10,24 The two most common missense amino acid substitutions in MUTYH result in reduced glycosylase activity and affinity for OG-containing substrates. 40 In addition, the corresponding E. coli variants are unable to distinguish OG from G. 25,36 This suggests that the function of MutY and its human homologue to prevent deleterious DNA mutations is critically dependent on the ability to detect OG and select the appropriate undamaged A for excision to initiate BER. …”

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confidence: 99%

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

et al. 2007

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Escherchia coli MutY plays an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG:A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG:A substrate on the kinetics of base removal, mismatch affinity and repair to G:C in an Escherchia coli-based assay. Surprisingly, adenine modification was tolerated in the cellular assay, while modification of OG results in minimal cellular repair. High affinity for the mismatch and efficient base removal require the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature for MutY to locate OG:A mismatches and select the appropriate adenines for excision to initiate repair in vivo prior to replication.The mismatch repair (MMR) pathway in E. coli relies on methylation at a GATC sequence to direct the repair machinery to remove the mismatched base on the newly synthesized strand. 1 However, almost two decades ago, an activity was detected in E. coli cell extracts that restored G:A mismatches to G:C matches and was insensitive to the methylation state of the template strand. [2][3][4] Concurrently, a mutator locus in E. coli (mutY) was identified that generated G:C → T:A transversion mutations. 5 It was later determined that the mutY gene product is an adenine glycosylase capable of removing adenines from G:A mismatches as the first step in base excision repair (BER). [6][7][8][9][10] The subsequent activity of downstream BER pathway enzymes, e.g. the AP endonuclease, deoxyribophosphate lyase, polymerase and ligase, restores the G:C base pair in a methylation-independent fashion. 11 MutY was also found to participate in the prevention of mutations caused by 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG, 1) by removal of adenine from OG:A mismatches. 10,12,13,14 Polymerase misinsertion of dAMP opposite OG creates OG:A mismatches that can lead to formation of T:A base pairs upon a second round of replication. 12 Recently, MutY has been in the spotlight due to the correlation between inherited biallelic mutations in the gene encoding the human homologue of MutY (MUTYH) and colorectal cancer. 10,15,16 *Author to whom correspondence should be addressed. Email: david@chem.ucdavis.edu,. # These authors contributed equally to this manuscript. We have previously examined the features of mismatched substrates that are required for efficient lesion recognition and adenine excision by MutY using pre-steady state and singleturnover kinetics. [17][18][19] Pre-steady state experiments revealed that MutY has a high affinity for the product such that release of the DNA product is rate-limiting. 17,19,20 Moreover, the identity of the base opposite A greatly affects both the rate of product release as well as the intrinsic rate of adenine removal determined under single-turnover conditions....

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Insight into the Roles of Tyrosine 82 and Glycine 253 in the Escherichia coli Adenine Glycosylase MutY

Cited by 38 publications

References 72 publications

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

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