Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY

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

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The oxidation product of 2′-deoxyguanosine, 7,8-dihydro-8-oxo- 2′-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2′-deoxy-2′-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.

Original languageEnglish (US)
Pages (from-to)14179-14190
Number of pages12
JournalBiochemistry
Volume44
Issue number43
DOIs
StatePublished - Nov 1 2005
Externally publishedYes

Fingerprint

Adenine
Glycine
Escherichia coli
Tyrosine
Mutation
Substrates
Viverridae
Geobacillus stearothermophilus
Deoxyguanosine
DNA
Bacilli
Enzymes
Amino Acid Substitution
Intercalation
Catalysis
DNA Replication
Leucine
DNA Repair
Colorectal Neoplasms
Repair

ASJC Scopus subject areas

  • Biochemistry

Cite this

Livingston, A. L., Kundu, S., Pozzi, M. H., Anderson, D. W., & David, S. S. (2005). Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY. Biochemistry, 44(43), 14179-14190. https://doi.org/10.1021/bi050976u

Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY. / Livingston, Alison L.; Kundu, Sucharita; Pozzi, Michelle Henderson; Anderson, David W.; David, Sheila S.

In: Biochemistry, Vol. 44, No. 43, 01.11.2005, p. 14179-14190.

Research output: Contribution to journalArticle

Livingston, AL, Kundu, S, Pozzi, MH, Anderson, DW & David, SS 2005, 'Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY', Biochemistry, vol. 44, no. 43, pp. 14179-14190. https://doi.org/10.1021/bi050976u
Livingston, Alison L. ; Kundu, Sucharita ; Pozzi, Michelle Henderson ; Anderson, David W. ; David, Sheila S. / Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY. In: Biochemistry. 2005 ; Vol. 44, No. 43. pp. 14179-14190.
@article{9e31fdf2d0b24576bbe142636e75fa1d,
title = "Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY",
abstract = "The oxidation product of 2′-deoxyguanosine, 7,8-dihydro-8-oxo- 2′-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2′-deoxy-2′-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.",
author = "Livingston, {Alison L.} and Sucharita Kundu and Pozzi, {Michelle Henderson} and Anderson, {David W.} and David, {Sheila S.}",
year = "2005",
month = "11",
day = "1",
doi = "10.1021/bi050976u",
language = "English (US)",
volume = "44",
pages = "14179--14190",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "43",

}

TY - JOUR

T1 - Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY

AU - Livingston, Alison L.

AU - Kundu, Sucharita

AU - Pozzi, Michelle Henderson

AU - Anderson, David W.

AU - David, Sheila S.

PY - 2005/11/1

Y1 - 2005/11/1

N2 - The oxidation product of 2′-deoxyguanosine, 7,8-dihydro-8-oxo- 2′-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2′-deoxy-2′-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.

AB - The oxidation product of 2′-deoxyguanosine, 7,8-dihydro-8-oxo- 2′-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2′-deoxy-2′-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.

UR - http://www.scopus.com/inward/record.url?scp=27444434257&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=27444434257&partnerID=8YFLogxK

U2 - 10.1021/bi050976u

DO - 10.1021/bi050976u

M3 - Article

VL - 44

SP - 14179

EP - 14190

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 43

ER -