ライフサイエンスコーパス: FAPy

1 ylamino-4(3H)-pyrimidinone 5'-monophosphate (FAPy).

2 ibosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy).

3 mino-4-hydroxy-5N-methylformamidopyrimidine (FaPy-7-MeGua), and abasic sites but not DNA substrates c

4 in duplex DNA the AFB moiety of the AFB-beta-FAPY adduct also intercalates on the 5' side of the pyri

5 The AFB-alpha-FAPY adduct blocks replication; it destabilizes the DNA

6 acking interactions induced by the AFB-alpha-FAPY adduct explain its lower stability as compared to t

7 Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two sepa

8 Herein, the structure of the AFB-alpha-FAPY adduct has been elucidated in 5'-d(C(1)T(2)A(3)T(4)

9 lower stability as compared to the AFB-beta-FAPY adduct in duplex DNA.

10 calation of the AFB moiety for the AFB-alpha-FAPY adduct in the tetramer 5'-d(C(1)T(2)X(3)A(4))-3', i

11 The FAPY adduct may be a major progenitor of aflatoxin B1-in

12 ken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicit

13 ide moiety is also observed for the AFB-beta-FAPY adduct, and suggests that the identity of the 3'-ne

14 The structure of a formamidopyrimidine (FAPY) adduct arising from imidazole ring opening of the

15 ive a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms.

16 rolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between alpha and beta

17 te and an AFB(1)-formamidopyrimidine (AFB(1)-FAPY) adduct.

18 type and supports conversion of GTP to both FAPy and APy.

19 imidazole ring opening [formamidopyrimidine (Fapy)] and is associated with significant myelosuppressi

20 ibosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass sp

21 orientation shifts to become parallel to the FAPY base and displaced toward the minor groove.

22 Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvert

23 NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now est

24 dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl

25 involving the R(a) axial conformation of the FAPY C5-N(5) bond and the E conformation of the formamid

26 pairs T(4).A(17) and X(5).C(16), placing the FAPY C5-N(5) bond in the R(a) axial conformation.

27 idopyrimidine nucleoside repair by examining Fapy*dA and Fapy*dG excision opposite all four native 2'

28 Fapy*dA is removed more rapidly than Fapy*dG, and duplex

29 tected from formamidopyrimidine nucleosides (Fapy*dA, Fapy*dG) via a pathway distinct from the Escher

30 st chemical syntheses of a monomeric form of Fapy-dA (1) and oligonucleotides containing this lesion

31 These results indicate that Fapy-dA and Fapy-dG will be sufficiently long-lived in D

32 es that the half-life for deglycosylation of Fapy-dA at 37 degrees C is approximately 103 h.

33 y 25 times more resistant to hydrolysis than Fapy-dA at 55 degrees C.

34 Deglycosylation of Fapy-dA in the monomer follows first-order kinetics from

35 The rate constants for deglycosylation of Fapy-dA in the monomeric and oligonucleotide substrates

36 Monomeric Fapy-dA readily epimerized at 25 degrees C in phosphate

37 containing the beta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides

38 Fpg efficiently excises Fapy.dA (K(m) = 1.2 nM, k(cat) = 0.12 min(-1)) opposite

39 ding lesions derived from 2'-deoxyadenosine, Fapy.dA and 8-oxo-dA, were not detectably mutagenic in t

40 The formamidopyrimidines Fapy.dA and Fapy.dG are produced in DNA as a result of o

41 as a tool to determine the configuration of Fapy.dA and Fapy.dG in DNA.

42 hibitor (K(I) = 3.5 +/- 0.3 nM) of repair of Fapy.dA by Fpg, suggesting the C-nucleoside may have use

43 These data suggest that Fapy.dA could be deleterious to the genome.

44 Incision of alpha-C-Fapy.dA follows Michaelis-Menten kinetics (K(m) = 144.0

45 Fapy.dA incision is considerably slower than that of alp

46 The extent of Fapy.dA incision suggests that the lesion exists predomi

47 Fapy.dA is produced in DNA as a result of oxidative stre

48 ng formamidopyrimidine lesions indicate that Fapy.dA is readily identified as an alkali-labile lesion

49 a higher frequency than 8-oxo-G-->T and that Fapy.dA is very weakly mutagenic, as is 8-oxo-dA.

50 ple turnovers are observed for the repair of Fapy.dA mispairs in a short period of time, indicating t

51 The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY i

52 Endo IV incision of Fapy.dA proceeds further upon rehybridization, suggestin

53 rachromosomal probes containing a Fapy.dG or Fapy.dA site-specifically incorporated, which showed une

54 IV incision of the C-nucleoside analogues of Fapy.dA was used to establish selectivity for the alpha-

55 e diastereomers of C-nucleoside analogues of Fapy.dA were introduced by using the respective phosphor

56 MutY also does not incise Fapy.dA when the lesion is opposite dG.

57 eta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides (K(D) < 27 nM),

58 , as well as the alpha-C-nucleoside (alpha-C-Fapy.dA) opposite dC (K(D) = 7.1 +/- 1.5 nM).

59 is considerably slower than that of alpha-C-Fapy.dA, and does not proceed to completion.

60 a duplex containing this nucleotide opposite Fapy.dA, nor does it exhibit an increased level of bindi

61 All duplexes containing Fapy.dA-dX or its C-nucleoside analogue melt lower than

62 entially cleaves duplex DNA containing alpha-Fapy.dA.

63 oved as efficiently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs.

64 ently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs.

65 A duplex containing a beta-C-Fapy.dA:T base pair is an effective inhibitor (K(I) = 3.

66 Fapy*dG (N6-(2-deoxy-alpha,beta-D-erythro-pentofuranosyl

67 Fapy*dG adopts the beta-anomer when base paired with cyt

68 s facile epimerization, but prior works with Fapy*dG analogues have precluded determining its effect

69 anscriptional bypass mutation frequencies of Fapy*dG and 8-OxodGuo measured in RNA products are compa

70 Promoter-dependent T7 RNAP bypass of Fapy*dG and 8-OxodGuo was carried out side by side.

71 ng the incorporation of nucleotides opposite Fapy*dG and potentially the repair of this structurally

72 Fitting of reaction data indicates that Fapy*dG anomers are kinetically distinguishable.

73 Extension from Fapy*dG at the 3'-terminus of a nascent primer is ineffi

74 Fapy*dG, inserts Fapy*dGTP, and extends from Fapy*dG at the primer terminus.

75 Primer-dependent transcriptional bypass of Fapy*dG by T7 RNAP is hindered compared to 2'-deoxyguano

76 ingle-nucleotide deletions are produced upon Fapy*dG bypass.

77 e nucleoside repair by examining Fapy*dA and Fapy*dG excision opposite all four native 2'-deoxyribonu

78 T7 RNAP incorporates cytidine opposite Fapy*dG in a miniscaffold at least 13-fold more rapidly

79 crystallographic characterization of natural Fapy*dG in duplex DNA and as the template base for DNA p

80 osure and increases the efficiency of beta-C-Fapy*dG insertion opposite dC.

81 A distinctive property of Fapy*dG is facile epimerization, but prior works with Fa

82 Fapy*dG is formed in greater amounts under anoxic condit

83 When Fapy*dG is in its nucleotide triphosphate form, Fapy*dGT

84 these data indicate that mutagenic bypass of Fapy*dG is likely to be the source of the mutagenic effe

85 e that T7 RNA polymerase (T7 RNAP) bypass of Fapy*dG is more complex than that of 8-OxodGuo.

86 Fapy*dG is more mutagenic in mammalian cells than 8-oxod

87 f single-stranded shuttle vectors containing Fapy*dG is more mutagenic than 8-OxodGuo.

88 eater error-prone Pol II bypass suggest that Fapy*dG is more mutagenic than 8-OxodGuo.

89 When Fapy*dG is present in the template, Pol beta incorporate

90 However, the molecular basis by which Fapy*dG is processed by DNA polymerases during this muta

91 Fapy*dG is unusual in that it exists as a dynamic mixtur

92 Extension of a nascent transcript past Fapy*dG is weakly dependent on the nucleotide opposite t

93 Determination of Fapy*dG mutagenicity in wild type and Pol beta knockdown

94 Fapy*dG preferentially gives rise to G -> T transversion

95 mentary kinetic studies have determined that Fapy*dG promotes mutagenesis by decreasing the catalytic

96 Much less is known about the effects of Fapy*dG than 8-OxodGuo on transcriptional bypass.

97 Formamidopyrimidine (Fapy*dG) is a major lesion arising from oxidation of dG

98 ,6-diamino-4-hydroxy-5-formamido pyrimidine (Fapy*dG) is a prevalent form of genomic DNA damage.

99 4,6-Diamino-5-formamidopyrimidine (Fapy*dG) is an abundant form of oxidative DNA damage tha

100 ,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy*dG) is formed from a common intermediate and in com

101 om formamidopyrimidine nucleosides (Fapy*dA, Fapy*dG) via a pathway distinct from the Escherichia col

102 Fapy*dA is removed more rapidly than Fapy*dG, and duplexes containing purine nucleotides oppo

103 ide soaking experiments trap the ring-opened Fapy*dG, demonstrating that ring opening and epimerizati

104 mammalian polymerase, bypasses a templating Fapy*dG, inserts Fapy*dGTP, and extends from Fapy*dG at

105 alytic efficiency of dCMP insertion opposite Fapy*dG, thus reducing polymerase fidelity.

106 omoter dependent RNA polymerase II bypass of Fapy*dG.

107 understanding of the promutagenic effects of Fapy*dG.

108 When the shuttle vector contains a Fapy*dG:dA base pair, as high as 20% point mutations and

109 For Fapy*dG:dA bypass, adenosine incorporation was greater t

110 Error-prone bypass of a Fapy*dG:dC base pair accounts for ~9% of the transcripts

111 d adenosine incorporation, particularly from Fapy*dG:dC bypass which yielded ~25% adenosine incorpora

112 (pol zeta) to incorporate an A opposite AFB1-Fapy-dG and extend from this mismatch, biological eviden

113 godeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol

114 d oligonucleotides containing this lesion or Fapy-dG at a defined site.

115 In COS7 cells, NM-Fapy-dG caused targeted mutations, predominantly G --> T

116 of the rate constant for deglycosylation of Fapy-dG in an oligonucleotide, revealed that this lesion

117 Although formation of NM-Fapy-dG in cellular DNA has been demonstrated, its poten

118 significant increases in the levels of AFB1-Fapy-dG in Neil1(-/-) vs. wild-type liver DNA.

119 NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals

120 Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation a

121 el synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotid

122 These results indicate that Fapy-dA and Fapy-dG will be sufficiently long-lived in DNA so as to

123 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-dG), could be useful in treating certain cancers.

124 -diamino-4-hydroxy-5-formyl amidopyrimidine (Fapy-dG), is associated with progression of age-related

125 N(5)-NM-substituted formamidopyrimidine (NM-Fapy-dG).

126 ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG).

127 ning yielding formamidopyrimidine AFB1 (AFB1-Fapy-dG).

128 uld catalyze high-fidelity synthesis past NM-Fapy-dG, but only on a template subpopulation, presumabl

129 To elucidate the mechanisms of bypass of NM-Fapy-dG, we performed replication assays in vitro with a

130 he alpha-anomer as a major contributor to NM-Fapy-dG-induced mutagenesis in primate cells.

131 tary d(CGCATGCGTACGC) counterpart yields two Fapy-dG.C duplexes that are differentially destabilized

132 Fpg excises Fapy.dG (K(M) = 2.0 nM, k(cat) = 0.14 min(-1)) opposite

133 Fapy.dG (N(6)()-(2-deoxy-alpha,beta-d-erythropentofurano

134 Fapy.dG and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-d

135 The similar effects of Fapy.dG and 8-oxodG on DNA polymerase and repair enzymes

136 a low dA misincorporation frequency opposite Fapy.dG and inefficient extension of a Fapy.dG:dA base p

137 Recently, Fapy.dG and its C-nucleoside analogue were incorporated

138 Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cell

139 MutY incises dA when it is opposite Fapy.dG and strongly binds duplexes containing the lesio

140 The formamidopyrimidines Fapy.dA and Fapy.dG are produced in DNA as a result of oxidative str

141 ee versus error-prone incorporation opposite Fapy.dG are significantly reduced in comparison with und

142 --> T transversion frequencies observed upon Fapy.dG bypass were <or=1.9% in wild-type E. coli.

143 These data demonstrate that Fapy.dG closely resembles the interactions of 8-oxodG wi

144 Fapy.dG containing dinucleotide phosphoramidites contain

145 , polymerase-mediated introduction of beta-C-Fapy.dG could be useful for incorporating useful amounts

146 es in vitro raise the question as to whether Fapy.dG elicits similar effects in vivo.

147 e alpha- and beta-configurational isomers of Fapy.dG have distinct effects on Pol II insertion and ex

148 Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --

149 o determine the configuration of Fapy.dA and Fapy.dG in DNA.

150 Replicative bypass of Fapy.dG in human cells is more mutagenic than that of 8-

151 Like 8-oxodG, Fapy.dG instructs DNA polymerase to misincorporate dA op

152 Overall, these experiments suggest that Fapy.dG is at most weakly mutagenic in E. coli.

153 Fapy.dG is formed in comparable yields under oxygen-defi

154 Under some conditions Fapy.dG is formed in greater yields than 8-oxodG from a

155 iperidine (1.0 M, 90 degrees C, 20 min), but Fapy.dG is less easily identified in this manner.

156 r in simian kidney (COS-7) cells showed that Fapy.dG is mutagenic inducing primarily targeted Fapy.G-

157 Fapy.dG is produced in DNA as a result of oxidative stre

158 Fapy.dG is produced in DNA as a result of oxidative stre

159 istic framework for better understanding how Fapy.dG lesions impact transcription and subsequent path

160 In contrast to OxodG bypass, Fapy.dG mutation frequencies were unaffected by carrying

161 incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is conv

162 The effect of Fapy.dG on replication in Escherichia coli was studied b

163 esis, there are no reports of the effects of Fapy.dG on RNA polymerase II (Pol II) activity.

164 kinetic studies to investigate the impact of Fapy.dG on three key transcriptional fidelity checkpoint

165 n using extrachromosomal probes containing a Fapy.dG or Fapy.dA site-specifically incorporated, which

166 ations observed above background when either Fapy.dG or OxodG was bypassed.

167 The interactions of DNA containing Fapy.dG or the nonhydrolyzable analogue with Fpg and Mut

168 different structures were solved, including Fapy.dG template-loading state (apo), error-free cytidin

169 Endo IV incises Fapy.dG to less than 5% under comparable reaction condit

170 for synthesizing oligonucleotides containing Fapy.dG utilized a reverse dinucleotide phosphoramidite,

171 the 5'-TGT sequence mutational frequency of Fapy.dG was approximately 30%, whereas in the 5'-TGA seq

172 Fapy.dG was bypassed less efficiently than OxodG.

173 anosine (8-OxodGuo) and formamidopyrimidine (Fapy.dG), are produced from a common chemical intermedia

174 uctural basis of transcription processing of Fapy.dG, five different structures were solved, includin

175 was found to be slightly less mutagenic than Fapy.dG, though it also exhibited a similar context effe

176 for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites

177 However, duplexes containing Fapy.dG-dA mispairs melt significantly higher than those

178 nds duplexes containing the lesion or beta-C-Fapy.dG.

179 Incision from Fapy.dG.dA is faster than from dG.dA mispairs but slower

180 ind duplexes containing Fapy.dG.dC or beta-C-Fapy.dG.dC compared to those in which the lesion is oppo

181 Fpg also prefers to bind duplexes containing Fapy.dG.dC or beta-C-Fapy.dG.dC compared to those in whi

182 Similarly, Fapy.dG:A mispair is extended with comparable efficiency

183 oduct state (postchemistry), and error-prone Fapy.dG:A product state (postchemistry), revealing disti

184 arable efficiency as that of the error-free, Fapy.dG:C base pair.

185 ATP binding state (prechemistry), error-free Fapy.dG:C product state (postchemistry), and error-prone

186 osite Fapy.dG and inefficient extension of a Fapy.dG:dA base pair work synergistically to minimize th

187 tal structures of a configurationally stable Fapy*dGTP analog, beta-C-Fapy*dGTP, with DNA polymerase

188 herefore, under oxidative stress conditions, Fapy*dGTP could become a pro-mutagenic substrate for ins

189 kinetic data indicate that binding of beta-C-Fapy*dGTP impedes enzyme closure, thus hindering inserti

190 ow DNA polymerase beta has evolved to hinder Fapy*dGTP insertion.

191 Kinetic analysis revealed that Fapy*dGTP is a poor substrate but is incorporated ~3-tim

192 ctive site residue, Asp276, positions beta-C-Fapy*dGTP so that it distorts the geometry of critical c

193 rase, bypasses a templating Fapy*dG, inserts Fapy*dGTP, and extends from Fapy*dG at the primer termin

194 y*dG is in its nucleotide triphosphate form, Fapy*dGTP, it is inefficiently cleansed from the nucleot

195 gurationally stable Fapy*dGTP analog, beta-C-Fapy*dGTP, with DNA polymerase beta.

196 the mutagenic effects of the lesion and not Fapy*dGTP.

197 polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less e

198 ses is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzy

199 cherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it di

200 efficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyro

201 ,6-diamino-4-hydroxy-5-f ormamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dG

202 .dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized.

203 cells if 1% of the dGTP pool is converted to Fapy.dGTP.

204 n 3-methyladenine DNA glycosylases I and II, FAPY DNA glycosylase, both known apurinic/apyrimidinic e

205 ichia coli, Fapy lesions are repaired by the Fapy-DNA glycosylase (Fpg) protein.

206 FAPY formation resulted in the loss of the guanine H8 pr

207 The FAPY formyl group was positioned to form a hydrogen bond

208 ed unequivocally that in simian kidney cells Fapy.G-->T substitutions occur at a higher frequency tha

209 .dG is mutagenic inducing primarily targeted Fapy.G-->T transversions.

210 Similarly, syn-Fapy.G:dATP pairing showed greater stacking in the 5'-TG

211 the 5'-TGA sequence, while stacking for anti-Fapy.G:dCTP pairs was similar in the two sequences.

212 Both Escherichia coli fapy glycosylase (Fpg) and human 8-oxo-DNA glycosylase (

213 ibosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), has been shown to require Mg2+ for catalytic acti

214 Gua, and (ii) one proposed rotamer of AFB(1)-FAPY is a block to replication, even when the efficient

215 AFB(1)-FAPY is detected at near maximal levels in rat DNA days

216 In Escherichia coli, Fapy lesions are repaired by the Fapy-DNA glycosylase (F

217 ucts allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from

218 Fapy lesions inhibit DNA synthesis likely modulating the

219 or exacerbating the mutagenic properties of Fapy lesions, their excision by three glycosylases, Fpg,

220 putatively nonmutagenic formamidopyrimidine (Fapy) lesions of adenine (Ade) and guanine (Gua) to eluc

221 putatively nonmutagenic formamidopyrimidine (Fapy) lesions.

222 events that permit GCH II to produce either FAPy or APy.

223 n became important when one of the two major FAPY species in DNA was found to be potently mutagenic a

224 In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers

225 C(16)A(17)T(18)A(19)G(20))-3' (X = AFB-alpha-FAPY) using molecular dynamics calculations restrained b

226 ng properties of this DNA adduct: (i) AFB(1)-FAPY was found to cause a G to T mutation frequency in E