ライフサイエンスコーパス: Pol epsilon

1 (pol beta), gamma (pol gamma), and epsilon (pol epsilon).

2 nd is significantly more accurate than yeast Pol epsilon.

3 nt the first report on the fidelity of human Pol epsilon.

4 it DNA polymerases: Pol alpha, Pol delta and Pol epsilon.

5 higher than that of a strain with wild-type Pol epsilon.

6 ion by proofreading-proficient pol delta and pol epsilon.

7 3' exonuclease of pol delta but not that of pol epsilon.

8 s homologous to the two smallest subunits of Pol epsilon.

9 esis by two major polymerases, pol delta and pol epsilon.

10 polymerases (pol): pol alpha, pol delta, and pol epsilon.

11 DNA pol epsilon and to full-length human DNA pol epsilon.

12 plays a critical role in the function of DNA pol epsilon.

13 ract with both p261 and p59 subunits of HeLa pol epsilon.

14 essential function of the C-terminal half of pol epsilon.

15 may be important for the normal functions of pol epsilon.

16 as pcna-90 was defective in replication with Pol epsilon.

17 TASOR mutants with reduced interactions with Pol epsilon.

18 rate pre-LC containing GINS, TOPBP1, and DNA pol epsilon.

19 of the DNA synthesis domain in the CMG-bound Pol epsilon.

20 tion by proofreading-proficient Pol delta or Pol epsilon.

21 a functional complex between CMG and native Pol epsilon.

22 pass, with a pattern most similar to that of Pol epsilon.

23 ly stimulate the activities of Pol alpha and Pol epsilon.

24 s 26% identity and 44% homology to the yeast pol epsilon 80-kDa subunit, DPB2.

25 Neither was Pol epsilon able to extend a D-loop in reconstitution ex

26 n Dbp2 directs leading ssDNA from CMG to the Pol epsilon active site.

27 ilon on the lagging strand, and CMG protects Pol epsilon against RFC inhibition on the leading strand

28 the reconstitution of the human four-subunit Pol epsilon and characterization of its catalytic proper

29 Direct binding between Pol epsilon and CMG provides an explanation for specific

30 racts with the leading-strand DNA polymerase Pol epsilon and contributes to the asymmetric segregatio

31 These findings distinguish Pol epsilon and delta functions in vivo and reveal tissu

32 -, and mismatch repair-mutant mice show that Pol epsilon and delta proofreading act in parallel pathw

33 Here we show that mouse Pol epsilon and delta proofreading suppress discrete mut

34 is hypothesized from these observations that pol epsilon and PCNA have separate but associated functi

35 Therefore, Pol epsilon and Pol alpha/delta seem to pursue their fun

36 eoprotein complexes, whereas in late S phase Pol epsilon and Pol alpha/delta were largely associated

37 ch repair (MMR) and replicative polymerases (Pol epsilon and Pol delta).

38 ing strand templates are primarily copied by Pol epsilon and Pol delta, respectively.

39 primarily replicated by two DNA polymerases, Pol epsilon and Pol delta, that function on the leading

40 es using Saccharomyces cerevisiae Pol delta, Pol epsilon and Pol eta and a series of matched and mism

41 ereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol epsilon and tethers it to the leading strand, and PC

42 we explored the genetic interaction between Pol epsilon and the main elements of the DNA damage resp

43 cations of antimutator amino-acid changes in Pol epsilon and their effects on mutation spectra sugges

44 mologous regions of both mouse and human DNA pol epsilon and to full-length human DNA pol epsilon.

45 he leading strand by DNA polymerase epsilon (Pol epsilon) and discontinuous synthesis of the lagging

46 catalytic subunit of DNA polymerase epsilon (pol epsilon) and that this enzyme is also required for D

47 , including alleles of pol alpha, pol delta, pol epsilon, and PCNA (proliferating cell nuclear antige

48 ollapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM

49 erating cell nuclear antigen, Pol-delta, and Pol-epsilon, and it contributes to the phosphorylation o

50 ve DNA polymerases Pol alpha, Pol delta, and Pol epsilon; and canonical maturation of Okazaki fragmen

51 was examined by indirect immunofluorescence, pol epsilon appeared in discrete nuclear foci that coloc

52 ity for frameshift errors observed for human Pol epsilon are distinct from the errors made by human P

53 Pol delta and pol epsilon are essential, but their roles in replicatio

54 SL/MYPS/N motif in region II in Pol delta or Pol epsilon are inviable.

55 ions in DNA polymerase catalytic residues of pol epsilon are lethal.

56 re N-terminal DNA polymerase domain of yeast pol epsilon are viable.

57 elta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are both required for efficient replication

58 antigen (PCNA), and DNA polymerase epsilon (Pol epsilon), are reduced at stalled replication forks.

59 elta, and epsilon (Pol alpha, Pol delta, and Pol epsilon), are responsible for eukaryotic genome dupl

60 A growing body of evidence specifies Pol epsilon as the leading strand DNA polymerase and Pol

61 the proofreading functions of pol delta and pol epsilon, as well as a temperature-sensitive pol epsi

62 ltiple permutations of the reconstituted CMG-Pol epsilon assembly.

63 We also show that the C terminus of Pol epsilon associates with origin DNA at the same time

64 lly lethal with pol2-M644G (encoding altered Pol epsilon base selectivity).

65 bulk of chromosomal DNA in eukaryotic cells, Pol epsilon being the main leading strand and Pol delta

66 -tier ahead of the C-tier places the leading Pol epsilon below CMG and Pol alpha-primase at the top o

67 nts of polymerase binding to CMG demonstrate Pol epsilon binds CMG with a Kd value of 12 nM, but Pol

68 pol alpha and pol delta, respectively), free pol epsilon binds single-stranded but not double-strande

69 Although wild type Pol delta and Pol epsilon both have high base substitution fidelity, P

70 This inhibition was observed for Pol epsilon but not with Pol delta, RB69 gp43 or Pol eta

71 gh as 40 micro M had no inhibitory effect on pol epsilon, but could inhibit pol alpha by 10-20% at 20

72 is supported only by the C-terminal half of pol epsilon, but it is disrupted in mutants affecting th

73 al and phylogenetically conserved subunit of pol epsilon, but not for interaction with DPB3.

74 y on these template-primers, indicating that pol epsilon, but not pol delta, can repair the incorpora

75 Consistently, Pol epsilon, but not Pol delta, was found in nuclear mat

76 These results demonstrate that Pol epsilon can act in eukaryotic MMR in vitro.

77 limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and

78 Contrary to previously characterized Pol epsilon cancer variants, the examined mutants cause

79 We find that human Pol epsilon carries out DNA synthesis with high fidelity

80 rts a model in which DNA polymerase epsilon (Pol epsilon) carries out the bulk of leading strand DNA

81 ta suggest that the examined domain supports Pol epsilon catalytic activity and symmetric movement of

82 Our work thus suggests a role of the Pol epsilon catalytic core in replisome formation, a rel

83 we report that a conserved domain within the Pol epsilon catalytic core influences both of these repl

84 olymerases (Pols), Pol alpha, Pol delta, and Pol epsilon, causing polymerase complex dissociation and

85 ission yeast cells lacking the N terminus of Pol epsilon (cdc20(DeltaN-term)) are hypersensitive to D

86 est that in the absence of the N terminus of Pol epsilon, cells accumulate DNA damage that must be re

87 DNA pol epsilon co-immunoprecipitated with MDM2 from transfe

88 on, we reconstituted a stable 15-subunit CMG-Pol epsilon complex and showed that it is a functional p

89 he purification, we were able to resolve the pol epsilon complex and truncated Pol2p (140 kDa), as wa

90 Here, we report reconstitution of the yeast pol epsilon complex, which was expressed and purified fr

91 at Dpb3 associates with other members of the Pol epsilon complex.

92 ficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and a

93 Pol epsilon contains two flexibly tethered lobes.

94 o repair the incorporated PMEG revealed that pol epsilon could elongate PMEG-terminated primers in bo

95 Incorporation of beta-L-Fd4CTP by pol epsilon could not be detected.

96 replication initiation, cdc20+ (encoding DNA Pol epsilon), cut5+ (homologous to DPB11/TopBP1), sna41+

97 e not observed with the exonuclease-inactive Pol epsilon-D290A,E292A variant lacking the catalytic re

98 ding yeast, we show that mutator variants of Pol epsilon depend on damage uninducible (Dun)1, an S-ph

99 Here we describe the reconstitution of Pol epsilon-dependent MMR using S. cerevisiae proteins.

100 e structure of the polymerase active site of Pol epsilon differs from those of other B family members

101 s 24 proteins, forming the CMG helicase, the Pol epsilon DNA polymerase, the RFC clamp loader, the PC

102 vation of the leading strand DNA polymerase, Pol epsilon, dNTP depletion, and chemical inhibition of

103 in fission yeast, and our data indicate that pol epsilon does not have a role as a checkpoint sensor

104 amino-terminal domain of the Dpb2 subunit of Pol epsilon (Dpb2NT) interacts with the Psf1 component o

105 roposed role in chromosomal replication, DNA pol epsilon (encoded by POL2) also functions directly as

106 ), indicating that high levels of unrepaired Pol epsilon errors drive extinction.

107 Also like pol alpha and pol beta, pol epsilon exhibits induced dNTP inhibition in the pres

108 Early in S phase, pol epsilon foci were adjacent to PCNA foci.

109 PCNA foci that were only present in S phase, pol epsilon foci were present throughout mitosis and the

110 Together, these data suggest that pol epsilon follows an ordered sequential ter-reactant m

111 delity, Pol delta is much less accurate than Pol epsilon for deletions involving repetitive sequences

112 he leading strand and is proposed to recruit Pol epsilon for leading-strand synthesis, but to date a

113 Exo1 substitutes for FEN1 and Pol epsilon for Pol delta with reasonable efficiency.

114 helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly.

115 the 5' end of the blocking primer inhibited Pol epsilon from synthesizing DNA up to the fork junctio

116 in contrast to both pol alpha and pol delta, pol epsilon functions more efficiently as gap size decre

117 prevalence of suppressors extragenic to the Pol epsilon gene suggests that factors in addition to pr

118 conditions used, we noted that depletion of Pol epsilon had a more pronounced inhibitory effect on c

119 Notably, we also discover that wild-type Pol epsilon has a sevenfold higher error rate when repli

120 Here, we show that RFC cannot load PCNA once Pol epsilon has been incorporated into the budding yeast

121 HeLa pol epsilon has been isolated by immunoaffinity purifica

122 Pol epsilon has been shown to play essential roles in ch

123 Whereas yeast Pol epsilon has been well characterized, human Pol epsil

124 Pol epsilon has greater contact with the nascent single-

125 from cells reduces BIR, whereas depletion of Pol epsilon has no effect.

126 to date a direct interaction between CMG and Pol epsilon has not been demonstrated.

127 synthesize DNA with high fidelity, but M644F Pol epsilon has reduced fidelity resulting from strongly

128 idence suggests that DNA polymerase epsilon (Pol epsilon) has a noncatalytic essential role during th

129 DNA polymerase epsilon (pol epsilon) has been implicated in DNA replication, DNA

130 man DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this p

131 However, the wild-type Pol epsilon holoenzyme efficiently displaced one nucleot

132 Human Pol epsilon holoenzyme is a monomeric complex containing

133 ma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly.

134 ve efforts, there is no atomic structure for Pol epsilon holoenzyme, critical to understanding how DN

135 The mutations affect the assembly of the pol epsilon holoenzyme.

136 Saccharomyces cerevisiae pol epsilon, however, consists of four subunits: a 256-k

137 and a potential tumor-suppressive effect of Pol epsilon in curbing genome re-arrangements.

138 cterization of the second largest subunit of Pol epsilon in fission yeast, called Dpb2.

139 ation is consistent with a specific role for pol epsilon in gap-filling in vivo.

140 en combined with recent evidence implicating Pol epsilon in leading strand replication, these data su

141 er, our results demonstrate that the role of Pol epsilon in replicative stress sensing is conserved i

142 also provide evidence for the direct role of Pol epsilon in replicative stress sensing.

143 itutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo

144 freading by 3' --> 5' exonuclease-proficient Pol epsilon, indicating that ribonucleotide incorporatio

145 We show here that the DNA pol epsilon-interacting domain of MDM2 is located betwee

146 veals its unexpected effect on incorporating Pol epsilon into the four-member pre-loading complex dur

147 e active site location occupied by Met644 in Pol epsilon is a key determinant of replication fidelity

148 These biochemical properties suggest that Pol epsilon is a likely source of ribonucleotides in hum

149 Hence, Pol epsilon is active with CMG on the leading strand, bu

150 Human Pol epsilon is also efficient at extending from primers

151 levels of dNTPs in vivo, and the activity of Pol epsilon is compromised more than lagging-strand poly

152 Interestingly, error specificity of M644F Pol epsilon is distinct from that of L868M/F Pol alpha o

153 placement synthesis by exonuclease-deficient Pol epsilon is distributive.

154 That Pol epsilon is essential for progression through S phase

155 ear antigen indicates that the reconstituted pol epsilon is functionally identical to native pol epsi

156 On its own, the Pol2 catalytic subunit of Pol epsilon is inefficient in CMG-dependent replication,

157 icating that ribonucleotide incorporation by Pol epsilon is likely a significant event in human cells

158 e observation that the essential function of Pol epsilon is not dependent on its DNA synthesis activi

159 ith other replicative DNA polymerases, human Pol epsilon is particularly accurate when copying homonu

160 We hypothesize that the C-terminal half of Pol epsilon is required for assembly of the replicative

161 f the vertebrate replisome that includes DNA Pol epsilon is retained on DNA.

162 bstitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreadi

163 many DNA transactions in vivo We found that Pol epsilon is unable to carry out extended strand displ

164 Our results suggest that Pol epsilon is unable to extend the invading strand in D

165 DNA polymerase epsilon (Pol epsilon) is a multi-subunit enzyme required for the

166 DNA polymerase epsilon (pol epsilon) is a multiple subunit complex consisting of

167 DNA polymerase epsilon (Pol epsilon) is a replicative DNA polymerase with an ass

168 DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic

169 catalytic subunit of DNA polymerase epsilon (Pol epsilon) is encoded by cdc20(+) and is essential for

170 haromyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for chromosomal replication.

171 DNA polymerase epsilon (Pol epsilon) is required for genome duplication and tumo

172 but addition of the Dpb2 protein subunit of Pol epsilon, known to bind the Psf1 protein subunit of C

173 Pol epsilon lacking 3' --> 5' exonuclease activity is le

174 e and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between t

175 emical evidence to support the proposal that pol epsilon may be dimeric in vivo.

176 point mutants suggest that the polymerase of pol epsilon may normally participate in DNA replication

177 n has suggested that DNA polymerase epsilon (Pol epsilon) may also play a role in MMR.

178 hat errors introduced by polymerase epsilon (Pol epsilon) may represent an alternative source of muta

179 n, suggesting that the small subunit of HeLa pol epsilon might be important for stability.

180 MCM, and the leading-strand DNA polymerase, Pol epsilon, move beyond the site of DNA synthesis, like

181 that the most common human cancer-associated Pol epsilon mutant (P286R) produces an excess of CpG>TpG

182 e conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta.

183 on), we report here on the fidelity of yeast Pol epsilon mutants with leucine, tryptophan or phenylal

184 epsilon, as well as a temperature-sensitive pol epsilon mutation, pol2-18, have no effect on tract s

185 ng cancer-associated DNA polymerase epsilon (Pol epsilon) mutation is a P286R substitution in the exo

186 Thus, dNTP pool levels correlate with Pol epsilon mutator severity, suggesting that treatments

187 Human DNA polymerase epsilon (pol epsilon) normally contains a 261-kDa catalytic subun

188 ciated with lamins, but in late S phase only Pol epsilon, not Pol alpha/delta, remained associated wi

189 r C (RFC) clamp loader specifically inhibits Pol epsilon on the lagging strand, and CMG protects Pol

190 primers on both strands that are extended by Pol epsilon on the leading strand and by Pol delta on th

191 tion between Dpb2 and CMG helps to stabilize Pol epsilon on the leading strand as part of a 15-subuni

192 g-strand synthesis, are observed when either Pol epsilon or Pol delta stalls at leading-strand damage

193 elity or the rates of DNA synthesis by human pol epsilon or the bacterial enzyme pol I.

194 e cloning of two additional subunits of HeLa pol epsilon, p17, and p12.

195 We show that the Pol epsilon p261 N-terminal catalytic domain is solely r

196 catalytic subunit with 39% identity to HeLa pol epsilon p261, a 80-kDa subunit (DPB2) with 26% ident

197 e crystal structure of the yeast orthologue, Pol epsilon-P301R, complexed with DNA and an incoming dN

198 luorescence in situ hybridization, the human pol epsilon p59 locus (DPE2) was assigned to chromosome

199 kDa subunit (DPB2) with 26% identity to HeLa pol epsilon p59, a 23-kDa subunit (DPB3), and a 22-kDa s

200 DNA polymerases zeta (pol zeta) and epsilon (pol epsilon) participated in AC > TT and A > T mutations

201 Our results support the hypothesis that Pol epsilon participates in short-patch base excision re

202 sociated functions early in S phase and that pol epsilon participates with PCNA in DNA replication la

203 ng and the DNA path through the CMG helicase-Pol epsilon-PCNA clamp.

204 In contrast to pol epsilon, pol delta exhibited negligible activity on

205 Intercrosses of Pol epsilon-, Pol delta-, and mismatch repair-mutant mic

206 the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase a

207 The Pol epsilon (pol2-Y831A) mutant is slightly sensitive to

208 plications of these findings for the role of Pol epsilon polymerase activity in DNA replication.

209 HeLa DNA polymerase epsilon (pol epsilon), possibly involved in both DNA replication

210 HeLa DNA polymerase epsilon (pol epsilon), possibly involved in both DNA replication

211 ethal relationship exists between defects in Pol epsilon proofreading (pol2-4) and MMR.

212 rring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair.

213 und to be significantly more pathogenic than Pol epsilon proofreading deficiency per se.

214 es the mutator phenotype of pol2-4 (encoding Pol epsilon proofreading deficiency) and is syntheticall

215 We found that inactivation of Pol epsilon proofreading elevates base-substitution muta

216 curate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution

217 epsilon is functionally identical to native pol epsilon purified from yeast and is therefore useful

218 A major form of pol epsilon purified from yeast consists of at least fou

219 140 kDa), as was observed initially with the pol epsilon purified from yeast.

220 logical concentrations of nucleotides, human Pol epsilon readily inserts and extends from incorporate

221 n MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR.

222 l epsilon has been well characterized, human Pol epsilon remains poorly understood.

223 cna-90 failed to interact with Pol delta and Pol epsilon, respectively.

224 e a 3.5- angstrom cryo-EM structure of yeast Pol epsilon revealing that the Dpb3-Dpb4 subunits bridge

225 c core in replisome formation, a reliance of Pol epsilon strand synthesis on a unique domain, and a p

226 Cells deficient in Pol epsilon subunits (POLE3 and POLE4) or the HUSH compl

227 since it does not bind to PCNA or the other pol epsilon subunits.

228 M644W and M644L Pol epsilon synthesize DNA with high fidelity, but M644F

229 Wild type Pol epsilon synthesizes DNA accurately, generating singl

230 e we demonstrate that the N-terminal half of Pol epsilon that includes the highly conserved polymeras

231 nce that MDM2 interacts with a region of DNA pol epsilon that plays a critical role in the function o

232 a eex mutants encoded second-site changes in Pol epsilon that reduced the pol2-4 mutator phenotype be

233 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling

234 ectrum of errors is similar to that of yeast Pol epsilon, there are several notable exceptions.

235 o assay to show that Dpb2NT is essential for Pol epsilon to interact with the replisome after initiat

236 Here, we explored the capacity of Pol epsilon to perform strand displacement synthesis, a

237 scent leading strand synthesis switches from Pol epsilon to Pol delta during replication termination.

238 nism whereby the HUSH complex functions with Pol epsilon to promote asymmetric H3K9me3 distribution a

239 Examination of the ability of pol delta and pol epsilon to repair the incorporated PMEG revealed tha

240 des an explanation for specific targeting of Pol epsilon to the leading strand and provides clear mec

241 lytic subunit of DNA polymerase epsilon (DNA pol epsilon), to a region that is known to be essential

242 ion, or by recruitment of Pol delta, but not Pol epsilon, to post-replicative processes such as trans

243 mutations affecting the polymerase domain of Pol epsilon trigger ATR-dependent signaling leading to S

244 plication, the DNA polymerases Pol delta and Pol epsilon utilise the ring-shaped sliding clamp PCNA t

245 When the subcellular localization of human pol epsilon was examined by indirect immunofluorescence,

246 sh2-Msh3), Exo1, RPA, RFC-Delta1N, PCNA, and Pol epsilon was found to catalyze an MMR reaction that r

247 sh2-Msh3), Exo1, RPA, RFC-Delta1N, PCNA, and Pol epsilon was found to catalyze both short-patch and l

248 NA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenes

249 DNA polymerase epsilon (pol epsilon) was purified to apparent homogeneity from h

250 The replicative DNA polymerase epsilon (Pol epsilon) was shown to activate the S-phase checkpoin

251 ions and fidelity of DNA polymerase epsilon (Pol epsilon), we report here on the fidelity of yeast Po

252 Using pure CMG and Pol epsilon, we reconstituted a stable 15-subunit CMG-Po

253 ical characterization of the interactions of pol epsilon with other replication, recombination, and r

254 the high-fidelity replicative pol delta and pol epsilon with the TLS pol theta, pol eta, Rev1, pol z