Research Article Association of DNA Repair Gene APE1...

Research ArticleAssociation of DNA Repair Gene APE1 Asp148GluPolymorphism with Breast Cancer Risk

Fatima AlMutairi1 Akbar Ali Khan Pathan12 Mohammed Alanazi1 Manal Shalaby13

Huda A Alabdulkarim4 Abdullah Alamri1 Abdulrahman Al Naeem5 Moammad Elrobh1

Jilani P Shaik1 Wajahatullah Khan6 Zahid Khan1 and Narasimha Reddy Parine1

1Genome Research Chair Department of Biochemistry College of Science King Saud University Riyadh 11451 Saudi Arabia2Integrated Gulf Biosystems Riyadh 11391 Saudi Arabia3Genetic Engineering and Biotechnology Research Institute (GEBRI) City for Scientific Research and Technology ApplicationsAlexandria 21934 Egypt4The Comprehensive Cancer Center King Fahad Medical City Riyadh 11525 Saudi Arabia5Department of Womenrsquos Imaging King Fahad Medical City Riyadh 11525 Saudi Arabia6Department of Basic Sciences College of Science and Health Professions King Saud Bin Abdul Aziz University for Health SciencesRiyadh 11426 Saudi Arabia

Correspondence should be addressed to Narasimha Reddy Parine reddyparinegmailcom

Received 13 February 2015 Revised 22 April 2015 Accepted 10 June 2015

Academic Editor Andreas Pich

Copyright copy 2015 Fatima AlMutairi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objective The aim of this study was to investigate the role of APE1 Asp148Glu polymorphism in breast cancer progression inSaudi populationMethods We examined the genetic variations (rs1130409) in the DNA base excision repair gene APE1 at codon148 (Asp148Glu) and its association with breast cancer risk using genotypic assays and in silico structural as well as functionalpredictions In silico structural analysis was performed with Asp148Glu allele and compared with the predicted native proteinstructure The wild and mutant 3D structures of APE1 were compared and analyzed using solvent accessibility models for proteinstability confirmation Results Genotypic analysis of APE1 (rs1130409) showed statistically significant association of Asp148Gluwith elevated susceptibility to breast cancerThe in silico analysis results indicated that the nsSNP Asp148Glu may cause changes inthe protein structure and is associated with breast cancer riskConclusion Taken together this is the first report that established thatAsp148Glu variant has structural and functional effect on the APE1 and may play an important role in breast cancer progression inSaudi population

1 Introduction

The incidence of breast cancer is highest among other cancersand is themain cause of cancer related deaths in Saudiwomen[1] Compared to developed and Western region nations theage adjusted rate for breast cancer in Saudi population is 4-5-fold lower however the median age of diagnosis is 49 yearswhich is significantly lower compared to Western patients[2 3] There is significant evidence that inadequate repairof DNA damage plays a major role in the progression ofcancer and other human diseases [4] Base excision repair(BER) is involved in repair of oxidative free radical induced

DNA lesions Efficiency of (BER) is suggested to be a majordeterminant of breast cancer risk [5] It is a key repair pathwaythat is accountable for conserving genome stability andconsequently protecting from cancer and other diseases byrepairing numerous lesions and strand breaks of DNA whichare uninterruptedly caused by endogenous and exogenousmutagens [6] When a single base is damaged the BER path-way enzymes are responsible for recognizing and repairingthe damaged base [7] The first step in base excision repairpathway uses DNA glycosylases to remove the damagedbase to form an abasic or AP site by cleaving the N-glycosylbond between the sugar and the base Following the removal

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 869512 10 pageshttpdxdoiorg1011552015869512

2 Disease Markers

of the damaged base apurinicapyrimidinic endonuclease 1(APE1) hydrolytically cleaves the phosphodiester backbone 51015840to the AP site resulting in the formation of a 51015840-deoxyribosephosphate (51015840-dRP) and a 31015840-OH primer [8] At this junctureDNA polymerase then inserts a correct nucleotide and DNAligases seal the repaired DNA strand APE1 is a multifunc-tional enzyme which is located on chromosome 14q112ndashq12 [9] It exhibits DNA repair activity and has a role inthe reductive activation of many transcription factors Thesetwo functions are encoded by two different sites of APE1enzyme The N-terminal region encodes the redox functionand the C-terminal region encodes the repair function [10]The DNA repair activity includes 31015840-phosphodiesterase 31015840-phosphatase and 3-51015840-exonuclease activities [11] To coordi-nate BER pathway APE1 interacts with PARP1 XRCC1 DNApolymerase 120573 and flap endonuclease 1 (FEN1) [12] APE1stimulates all these proteins individually DNA polymerasethen inserts a correct nucleotide and DNA ligases seal therepaired DNA strand Mutations in this highly regulatedmechanism can be caused by single nucleotide polymor-phisms (SNPs) which will result in insufficient DNA repairwhich may enhance DNA lesions [13 14] Mutations in APE1gene may affect its function If AP sites are not processedmutagenesis and cellular cytotoxicity can result from blockedDNA replication machinery or from misincorporation ofbases opposite theAP site [12 15]TheAPE1 polymorphism atcodon 148 has been previously reported to be associated withprostate cancer risk [14] However previous reports on theassociation between APE1 polymorphisms and cancer riskhave provided inconsistent results [16ndash19] Data pertainingthe association of the APE1 polymorphisms with breastcancer risk are also inconsistent [4 20] To the best of ourknowledge there is no published report on the associationbetweenAPE1 SNP variant Asp148Glu (rs1130409) and breastcancer in Saudi population Thus this is the first study thatinvestigates APE1 SNP Asp148Glu association with breastcancers using TaqMan assay in Saudi women Additionallyin silico analyses were performed to determine the structuraland functional consequence of Glu instead of Asp at codon148 of APE1 protein

2 Materials and Methods

21 Study Population This study was a case-control studythat included 100 Saudi female patients diagnosed with breastcancer at King Fahad Medical City Hospital Riyadh SaudiArabia along with 100 controls Controls were age-matchedand confirmed to be free of cancer and other diseases follow-ing thorough physical examinations The demographic dataof each patient and control were recorded (Table 1) Patientsand controls were enrolled under King Khalid UniversityHospital Institutional Review Board approved protocol withwritten informed consent

22 Genotyping Genomic DNA was extracted from theblood samples of breast cancer cases and controls usingQIAmp DNA blood Mini Kit (Qiagen Valencia CA) follow-ing the manufacturerrsquos instructions SNP rs1130409 in APE1gene was genotyped using TaqMan allelic discriminationassay as described previously [21 22] Ten percent of the

Table 1 Clinical characteristics of study subjects

Variable Character Number of samplesAge (years)Median age (48 plusmn 82lowast) lt48gt48 4753

Estrogen receptor ER+ERminus 5545Progesterone receptor PR+PRminus 5644HER status HER+HERminus 4057lowastMedian age with standard deviation

samples were subjected to repeated analysis for verificationof genotyping procedures

23Modeling ofMutant Structure X-ray diffraction structureof APE1 (2O3H) from PDB database was used as a referenceto compare the wild-type (Asp148) protein structure withthe predicted mutant structure (148Glu) and its solventaccessibility including secondary structures was modeledusingmolecular dynamics (MD) simulationThe best homol-ogy model for APE1 protein with Asp148Glu was selectedusing I-TASSER server [23] The mutant APE1 Asp148Glu3-D structure was predicted using Modeller 9v10 [24] Thepredicted model for mutant type protein was evaluated usingProSA-web [25]

24 Analyzing the Effects of Mutation on Protein StabilityStability of the mutant protein is checked using prediction ofPrediction of Protein Mutant Stability Changes (PoPMuSiC)[26] The results were based on the selected ΔΔ119866 values inkcalmol of the predictedAPE1Asp148Glu structure to evalu-ate the change in folding free energy after mutation (ΔΔ119866)Additionally CUPSAT (Cologne University Protein StabilityAnalysis Tool) was also used to confirm the results [27]

25 The Effect of Mutant Residue 148Glu on APE1 Structureand Function The presence of glutamate instead of aspartateat codon 148 of APE1 may affect the overall structure with apotential to alter its activityThe effect of Asp148Glumutationwas analyzed using Have yOur Protein Explained (HOPE)program [28] as described earlier by Alanazi et al [29]

26 Molecular Dynamics Simulation The effect of glutamateat codon 148 on APE1 was studied by comparing with three-dimensional structure of APE1 protein present in the proteindatabank [PDB 2O3H] Structures deduced forAPE1 harbor-ingmutationAsp148Gluwhich is identified to be risk in Saudibreast cancer samples were utilized for the analyses Biopoly-mer module in InsightII was used to substitute aspartate withglutamate residue at codon 148 from the fragment library andto addhydrogen atoms to bothwild-type andmutated proteinstructures at pH 70 (Accelrys Inc San Diego CA) Themolecular simulation programCHARMMwas used to derivethe force fields of both structures [30] A sequence of energyminimization steps were performed as described by Alanaziet al [31] on native and mutant protein structures by usingInsightIIDiscover (Accelrys Inc San Diego CA) Followingenergy minimization protein structures were analyzed usingDiscovery Studio 25 (Accelrys Inc San Diego CA)

Disease Markers 3

Table 2 Genotype distribution of APE1 gene polymorphism in breast cancer cases and controls

Genotype Cases Controls OR 95 CI 1205942 119901 valuers1130409AspAsp 12 (012) 27 (027) RefAspGlu 45 (045) 46 (046) 2201 0994ndash4872 387 004917GluGlu 43 (043) 27 (027) 3583 1558ndash8243 942 000215AspGlu + GluGlu 88 (088) 73 (073) 2712 1284ndash5727 717 000743Asp 69 (0345) 100 (05) RefGlu 131 (065) 100 (05) 1899 1270ndash2839 985 000170

Table 3 Genotype frequencies of APE1 gene polymorphism in below 48 and above 48 years of age of breast cancer cases and controls

Genotype Cases Controls OR 95 CI 1205942 119901 valuers1130409 Below 48AspAsp 7 (0148) 16 (0275) RefAspGlu 24 (0510) 26 (0448) 2110 0740ndash6013 199 015841GluGlu 16 (0340) 16 (0275) 2286 0741ndash7051 211 014678AspGlu + GluGlu 40 (0851) 42 (0724) 2177 0810ndash5847 244 011790Asp 38 (0404) 58 (05) RefGlu 56 (0595) 58 (05) 1474 0851ndash2553 192 016607rs1130409 Above 48AspAsp 5 (0094) 11 (0261) RefAspGlu 21 (0396) 20 (0476) 2310 068ndash784 185 017378GluGlu 27 (0509) 11 (0261) 5400 152ndash1920 739 000656AspGlu + GluGlu 48 (0905) 31 (0738) 3406 108ndash1075 470 003021Asp 31 (0292) 42 (05) RefGlu 75 (0707) 42 (05) 2419 133ndash440 853 000349

27 Statistical Analysis Genotype and allelic frequencieswere compared using Fisherrsquos exact test (two-tailed) asdescribed by Alanazi et al [21] to estimate the 1205942 testand odds ratios (OR) and 95 confidence intervals (CI) toknow the variation between cancer cases and controls Allstatistical analyses were performed using Statistical Packagefor the Social Sciences version 210 (SPSS Inc Chicago IL)The allele and genotype frequencies of APE1 (rs1130409)polymorphisms in the central region population of SaudiArabia (CRS) were compared with some of the populationsof the HapMap database for example Utah residents withnorthern and western European ancestry from the CEPHcollection (CEU) Han Chinese in Beijing China (CHB)Yoruba in Ibadan Nigeria (YRI) Maasai in Kinyawa Kenya(MKK) Japanese in Tokyo Japan (JPT) Gujarati Indians inHouston Texas (GIH) andToscans in Italy (TSI) as describedpreviously by Alanazi et al [32] Pairwise Chi-square (1205942)tests were performed between the central region populationof Saudi Arabia (CRS) and other populations using the allelefrequencies in a 2 times 2 contingency table to study if thecentral region of Saudi population (CRS) shows significantdifferences compared to other populations

3 Results

The present study examined the SNP rs1130409 (Asp148Glu)of APE1 gene in a total of 200 subjects The distribution ofthe three genotypes as AspAsp AspGlu and GluGlu at

codon 148 of the APE1 was significantly different between thecontrols and breast cancer patients (1205942 = 944 df = 2 and119901 = 00089) The genotype frequencies in breast cancer caseswere 012 045 and 043 for AspAsp AspGlu and GluGlurespectively whereas in healthy controls the frequenciesof AspAsp AspGlu and GluGlu were 027 046 and027 respectively (Table 2) The heterozygotes (AspGlu) andhomozygote variant (GluGlu) showed significantly higherrisk in breast cancer patients compared with the controls(AspGlu OR = 220 1205942 = 387 and 119901 = 00491 GluGluOR 358 1205942 = 942 and 119901 = 00021) The Glu allelicfrequency of rs1130409was higher (0655) in the breast cancerpatients than that in the control group (050) (OR = 1891205942 = 985 and 119901 = 00017) (Table 2)

31 Effect of Age on the Association of APE1 SNP Asp148Gluwith Breast Cancer To examine the association of the SNPswith the age at the time of breast cancer diagnosis we strat-ified the patients according to the median age at diagnosis(Table 1) as le48 (119899 = 47) or gt48 (119899 = 53) years andcompared them with age-matched controls The analysesshowed that the SNP rs1130409 did not have any associationwith breast cancers arising in women at or below 48 yearsof age (Table 3) However APE1 codon 148 variant showedsignificant association with elderly breast cancer patients(gt48 years) withGluGlu genotype aswell asGlu allele posinghigher risk (Table 3)

4 Disease Markers

Table 4 Genotype frequencies of APE1 gene polymorphism in breast cancer cases ER

Genotype Cases Controls OR 95 CI 1205942 119901 valuers1130409 ER +veAspAsp 7 (0127) 27 (027) RefAspGlu 23 (0418) 46 (046) 1929 0731ndash5089 179 018065GluGlu 25 (0454) 27 (027) 3571 1322ndash9645 665 000992AspGlu + GluGlu 48 (0872) 73 (073) 2536 1023ndash6286 422 003992Asp 37 (0336) 100 (05) RefGlu 73 (0663) 100 (05) 1973 1217ndash3198 771 000551rs1130409 ER minusveAspAsp 5 (0111) 27 (027) RefAspGlu 22 (0488) 46 (046) 2583 0876ndash7613 309 007881GluGlu 18 (04) 27 (027) 3600 1168ndash1109 530 002127AspGlu + GluGlu 40 (0888) 73 (073) 2959 1057ndash8281 456 003281Asp 32 (0355) 100 (05) RefGlu 58 (0644) 100 (05) 1812 1085ndash3027 522 002230

Table 5 Allele and genotype frequencies of APE1 rs1130409 (Asp148Glu) in central Saudi Arabia and other populations

PopulationGenotype freq (number) Allele frequency Pairwise 1205942 test value

between CRS amp otherpopulations

119901 valueAspAsp freq(number)

AspGlu freq(number)

GluGlu freq(number)

Wild typeAsp

VariantGlu

CEU (119899 = 226) 0319 0407 0274 052 048 013 071CHB (119899 = 84) 0214 0476 0310 045 055 041 051JPT (119899 = 170) 0094 0494 0412 034 066 661 001GIH (119899 = 176) 0114 0341 0545 028 072 1286 00003lowast

YRI (119899 = 226) 0133 0381 0487 032 068 924 0002lowast

MKK (119899 = 286) 0098 0524 0378 036 064 605 001TSI (119899 = 174) 0230 0448 0322 045 055 053 046CRS (119899 = 100) 027 046 027 050 050 Ref mdashPopulation descriptionsCEU Utah residents with Northern and Western European ancestry from the CEPH collectionCHB Han Chinese in Beijing ChinaJPT Japanese in Tokyo JapanGIH Gujarati Indians in Houston TexasYRI Yoruba in Ibadan NigeriaMKK Maasai in Kinyawa KenyaTSI Toscans in ItalyCRS Saudi population residing in Riyadh region of central Saudi Arabialowast119901 values significant after Bonferroni correction

32 Effect of ER Status on the Association of APE1 SNPAsp148Glu with Breast Cancer The association of breastcancer risk with the SNP variant rs1130409 based on theestrogen receptor (ER) status of the cancer patients was alsoinvestigated The genotype distribution in the ER+ (119899 = 55)and ERminus (119899 = 45) was independently compared with thegenotypes in the control samples (119899 = 100) (Table 4) APE1variant rs1130409 showed higher risk in both ER+ and ERminusbreast cancer patients for GluGlu genotype and Glu alleles(Table 4)

33 Genotype and Allele Frequencies of APE1 Variantrs1130409 in Saudis andOther Populations We compared thegenotypic and allelic frequencies of the APE1 SNP rs1130409in a normal healthy Saudi population with some other

populations available in the HapMap database The observedAspAsp AspGlu and GluGlu genotype frequencies inthe Saudi population were 027 046 and 027 respectively(Table 5) Both the Asp (wild-type) and the Glu (variant)allele frequencies were similar and found to be 050 in Saudipopulation The variant allele frequency ranged from 048in CEU to 072 inGIH population The CRS populationfrequencies differed significantly from GIH YRI and MKKpopulations based on the pairwise Chi-square (1205942) tests(Table 5)

34 Effect of APE1 Asp148Glu Substitution on Protein Sta-bility Thermodynamic protein stability variations due tothe replacement of Asp148 with 148Glu in the APE1 werepredicted using PoPMusic It revealed that the amino acid

Disease Markers 5

HO

O

OH

O

148

OH

O

OH

O

Aspartic acid Glutamic acid

H2NH2N

Mutates to Overlap

Figure 1 Schematic structure of the wild-type and mutant (Asp148Glu) amino acid

alteration resulted in low levels of folding free energy (ΔΔ119866 =116 kcalmol) and caused structural destabilizing effectsSimilar results were observed with CUPSAT as well APE1Asp148Glu exhibited unfavorable changes in torsion angleswhich influenced the overall stability of the protein APE1148Glu tertiary structure revealed significant variations dueto protein folding in the mutated region between predictedand measured stability changes

35 Effect of Mutant Residue 148Glu on APE1 Structure andFunction The mutant Glu residue at position 148 of APE1proteinwas bigger than thewild-typeAsp residueHave yOurProtein Explained (HOPE) which collects and combinesinformation from several webservers and databases was usedto analyze the effect of Asp148Glu on APE1

DomainsAsp148Glu residue is part of APE1 interpro domainnamed ldquoExodeoxyribonuclease III xthrdquo (IPR004808 seehttpwwwebiacukinterproentryIPR004808jsessionid=BEC787876CD492333C5F42D24F5AFD6B) This domain isannotated with the following Gene-Ontology (GO) termsto indicate its function nuclease activity (GO 0004518)and DNA repair (GO 0006281 see httpwwwebiacukQuickGOGTermid=GO0006281) More broadly speakingthese Gene-Ontology annotations indicate that the domainhas a function in hydrolase activity (GO 0016787 see httpwwwebiacukQuickGOGTermid=GO0016787)This res-idue is part of an interpro domain named ldquoEndonucleaseexonucleasephosphataserdquo (IPR005135 see httpwwwebiacukinterproentryIPR005135jsessionid=05F95A5D842E-BA0A1048B8D753BB9705) Since the mutant residue waslocated in a domain which plays an important role primarilyin the protein activity it might disturb this function

Conservation The wild-type amino acid was not conservedat position 148 of APE1 protein and another residue type wasobserved more often at this position in other homologoussequences This means that other homologous proteins existwith that other residue type more often than with the wild-type residue in the protein sequenceTherefore the mutationis possibly damaging

AminoAcid PropertiesThewild andmutant type amino acidsdiffer in size where themutant residuewas found to be biggerwhich may lead to altered structure

36 MD Simulations of the Wild-Type and Mutant APE1The 3D structure of APE1 (PDB ID 2O3H) was alreadyavailable from the protein database This structure was usedto examine the structural and functional effects of Asp148Glusubstitutions in APE1 The structures of the wild-type aminoacid aspartate and the risk conferring mutant amino acidglutamate were studied (Figure 1) The backbone was thesame for the wild-type and the variant structures (shownin red color) whereas the side chain which was unique forwild and mutant type is shown in black color (Figure 1)Each amino acid depending on its side chain has its ownspecific size charge and hydrophobicity values The mutantresidue (Glu) due to the presence of an additional methylenegroup was larger in size than the wild-type (Asp) residueWhen compared with other homologous protein sequencesthe presence of the mutant glutamate reside at its respectiveposition was not found to be conserved and hence couldalter the structure and may have deleterious effect on APE1function

Substitution of Asp148 with 148Glu resulted in a slightworsening of ProSA-web 119885-score from minus513 to minus813(Figure 2)The total energy deviation was minus3 whichmay havea very unfavorable effect on the APE1 protein structure andfunction

Molecular dynamics simulations were carried out usingthe APE1 structural information from the PDB databaseTheamino acid sequence and the open reading frame of the APE1were submitted to I-TASSER program and of the best fivemodels that were generated 2O3H was selected based onthe 119862-score (minus092) TM-score (060 plusmn 014) RMSD (84 plusmn45 A) number of decoys (4094) and cluster density (00756)The human APE1 (PDB ID 2O3H) has 285 amino acidresidues with three side chains (A B and C) (Figure 3(a))The predicted structure with altered APE1 variant Asp148Gluwas studied using Discovery Studio 25 and compared withthe native structure (Figure 3(b)) The target amino acid atposition 148 of APE1 protein was mutated from Asp to Gluand selected for the lowest energy rotamer conformationsThe lowest potential energy state was achieved by atomicposition arrangements using Steepest Descent (SD) energyminimization protocol for 200 steps and all water moleculeswere subsequently removed from the resulting structureTheParticle Mesh Ewald summation method was used for theestimation of the electrostatic energy with a distance cutoff

6 Disease Markers

Number of residues0 200 400 600 800 1000

X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5minus513

minus813

Figure 2 Stability change of the mutant calculated by ProSA server

of 10 A Similar procedure was followed for wild-type APE1(PDB ID 2O3H) structure to relax the crystal packing forceto compare it with themutant structure Consequent to salva-tion the resultant solvate showed successful accumulation ofsolvent around the predicted structureTheoctahedral shapesof water box fitted fully to solvate the APE1 proteinmoleculeswith an edge distance of 100 A (Figure 3(c)) The wild andthe mutant structures were superimposed to detect the effectof structural changes due to the mutation (Figure 3(b)) Thestructural and functional studies suggest that the variantallele (Asp148Glu) was localized in APE1 binding regionhence the mutation may play a significant role by alteringits binding efficacy to its substrate and thus affecting thestructural and functional properties of protein

4 Discussion and Conclusions

The apurinicapyrimidinic endonuclease (APE) APE1 isinvolved in the BER pathway [33] Gene encoding APE1has five exons with a 221 kb coverage on chromosome 14(14q112ndashq12) when hydrolysed at the 31015840 end it blocks DNAoxidisation thus producing 31015840-hydroxyl termini which isrequired for DNA repair during single- or double-strandbreaks [34 35] Imbalances in this tightly regulated processdue to SNP may cause insufficient DNA repair mechanismand accumulate DNA breaks In the present study we exam-ined the role of APE1 variant Asp148Glu and breast cancerrisk in Saudi females The results showed that Asp148Gluvariation may increase the risk of breast cancer by approxi-mately 35-fold in Saudi patients (Table 2) Furthermore theresults also indicate that the Asp148Glu polymorphism was

also associated with increased risk of breast cancer amongsubgroups of older subjects (gt48 years) in ER positive groupas well as ER negative group (Tables 3 and 4) It is possiblethat the older individuals who showed higher risk associationwith breast cancer were more likely due to aging rather thandirect genetic effects It is more plausible that alteration in theAPE1 gene may be more influential in early onset of breastcancer however such an association was not observed in ouryounger group of patients (age le 48 years) probably due tosmall sample size

This is the first report that deals with the APE1 variationAsp148Glu which significantly contributes to breast cancersusceptibility in Saudi females and suggests the importanceof APE1 in breast carcinogenesis The elevated risk of breastcancer in subjects with the APE1 alteration (Asp148Glu)can be attributed to the reduced APE1 activity in the DNAbase excision repair pathway Recent meta-analysis study[36] suggests that Asian populations are at higher risk ofdeveloping cancer than the non-Asian populationswithAPE1Asp148Glu variant Our results are in agreement with thisobservation and confirm that the Glu residue at position 148of the APE1 confers significantly higher risk of breast cancerin Saudi females

Saudi Arabian population has various tribes settled indifferent provinces for decades and these are usually recog-nized by their family names The families residing in variousprovinces have been clustered based on their origin [32 37]In the present study the genotype and allele frequenciesof rs1130409 (Asp148Glu) in a central region populationof Saudi Arabia were observed and compared with variouspopulations of HapMap database The results showed that

Disease Markers 7

MD simulation spherical solvent boundary potential

Trun

cate

d oc

tahe

dron

bou

ndar

y ex

plic

it w

ater

solv

ated

syste

m 5

700

wat

er m

olec

ules

and

solv

ate

Chain A

Chain B Chain C

Chain A

051M ionKCI ion (simulation)

prot

ein

with

edge

dist

ance

10Aring

cuto

ff

Asp148

Structure superimposed with wild and mutant residues

Asp148 = Glu

Loop10

Asp148=

Glu

(a)

(b)

(c)

Figure 3 (a) Ribbon diagram of APE1 protein showing all the three side chains A B and C (b) APE1 structure superimposed with wild-typeand mutant residue with mutation Asp148Glu enlarged (c) Molecular dynamics (MD) simulation showing truncated octahedron boundaryexplicit water solvated and hydrogen atomsThe visual inspection also allows identifying the side chain of Glu residues involved in hydrogenbonding with the surrounding molecules

the allelic frequencies for rs1130409 (Asp148Glu) were signif-icantly different in the Saudi population compared to GIHYRI and MKK populations of HapMap database (Table 5)However Chinese Japanese Italian and northwestern Euro-pean populations showed no significant difference in allelicfrequencies for rs1130409 variant compared to the Saudicentral region population Hence examining the SNP variantin other populations probably will not yield similar resultsalthough APE1 (Asp148Glu) has previously been reported tobe associated with breast cancer risk in Asian and Europeanpopulations based on meta-analysis results

We also evaluated the effect of Asp148Glu mutationon APE1 protein structure Molecular dynamics methodsusing simulations in explicit solvent conditions were appliedfor investigating the wild and mutant amino acids andvariation in APE1 protein dynamics and stability due toAsp148Glu variationThe energy minimization studies of thewild-type protein (Asp148) and the mutant type (148Glu)structures revealed that there was energy deviation dueto Asp148Glu mutation (minus3 kcalmol) The mutant APE1(Asp148Glu) structure stability based on thermodynamicchanges was also detected using linear mixture of statisticalpotentials Protein stability estimation using PoPMusic and

CUPSAT revealed that variant Asp148Glu caused structuraldestabilizing effects on the APE1 protein structure Structuraland functional analysis of the wild-type and variant APE1revealed numerous multimer contacts including the oneassociated with nuclease (GO 0004518) and hydrolase activ-ity (GO 0016787) APE1 variant Asp148Glu was present inan interpro domain exonucleaseendonucleasephosphatase(IPR005135) which is responsible for the main activity of theprotein therefore any mutation in this region may affect thefunction of the protein

Along with DNA repair activity APE1 has another majorfunction which is also known as the redox effector factor1 (Ref-1) [11] APE1Ref-1 reductively activates transcriptionfactors including c-Jun activator protein-1 (AP-1) nuclearfactor kappa B (NF-120581B) the tumor-suppressor protein p53hypoxia-inducible factor 1a (HIF-1a) and paired box gene 8which are involved in various cellular processes such as cellsurvival growth signaling and inflammatory pathways [38ndash41] APE1 was also identified as a direct trans-acting factor forrepressing genes by binding to the negative calcium-responseelement in their promoters APE1Ref-1 dysregulation hasbeen reported to be associated with several diseases suchas neurodegenerative [42] and cardiovascular diseases [43]

8 Disease Markers

and with various human cancers [44 45] APE1Ref-1 mayhave a role in cancer progression via its ability to increaseDNA repair and antiapoptotic inflammatory and growth-promoting activities [46] APE1 gene polymorphisms maylead to amino acid substitutions which may result in alter-ations of the functions of APE1Ref-1146 Our results supportmost of the previous studies which stated that Asp148Glu(TG codon 148 exon 5 and Asp to Glu) has a role in cancerdevelopment and progression APE1 Asp148Glu mutation isreported to be found among different populations with highfrequency and has been associated with various tumors [47ndash49]

Overall our study has several key findings based ongenetic and computational methods to implicate APE1 in thedevelopment of breast cancer The strength of this study isthat cancer cases and normal control samples were collectedfrom the central region of Saudi Arabia and errors ingenotyping were evaded by replicating select samples forrandom confirmation of the results Limitations of theseassociation analyses include the fact that the breast cancercases were stratified for certain variables such as age at cancerdiagnosis and ER status to assess its possible effect howeverthe sample size is small and limited to the central region ofSaudi population Hence in future studies the present datashould be validated with larger number of samples as well asin other ethnic groups living in Saudi Arabia

In conclusion this is the first study showing an associa-tion between the APE1 Asp148Glu genotypes and increasedrisk of breast cancer in Saudi patients Genotyping and insilico prediction based on MD simulation results suggest thatthe APE1 Asp148Glu variant may alter the BER pathwayactivity hence probably contributing to breast carcinogenesisas its dysfunction may play a major role in the developmentof breast carcinoma Additional detailed functional as wellas association studies with larger sample size are neededto elucidate the role of APE1 polymorphism and associatedbreast cancer risk in Saudi population

Abbreviations

APE1 Apurinicapyrimidinic endonuclease 1MD simulation Molecular dynamics simulationPoPMuSiC Prediction of Protein Mutant Stability

ChangesCUPSAT Cologne University Protein Stability

Analysis ToolHOPE Have yOur Protein Explained

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Narasimha Reddy Parine Mohammed Alanazi and ManalShalaby conceived and designed the experiments FatimaAlMutairi Akbar Ali Khan Pathan Narasimha Reddy Parineand Jilani P Shaik performed the experiments Narasimha

Reddy Parine Akbar Ali Khan Pathan Fatima AlMu-tairi and Wajahatullah Khan analyzed the data NarasimhaReddy Parine Zahid Khan Wajahatullah Khan MohammedAlanazi and Fatima AlMutairi wrote the paper Sample col-lection and clinicopathological data were performed byHudaA Alabdulkarim and Abdulrahman Al Naeem NarasimhaReddy Parine FatimaAlMutairi Akbar Ali Khan Pathan andMohammed Alanazi interpreted the data Fatima AlMutairiand Akbar Ali Khan Pathan contributed equally

Acknowledgment

This project was funded by the National Plan for ScienceTechnology and Innovation (MAARIFAH) King AbdulazizCity for Science and Technology Kingdom of Saudi ArabiaAward no 12-MED2622-02

References

[1] J Ferlay I Soerjomataram M Ervik et al ldquoGLOBOCAN 2012v10 cancer incidence and mortality worldwiderdquo IARC CancerBase 11 International Agency for Research on Cancer LyonFrance 2013 httpglobocaniarcfr

[2] Saudi National Cancer Registry Cancer Incidence and SurvivalReport Saudi Arabia 2007 Saudi National Cancer RegistryRiyadh Saudi Arabia 2011

[3] W F Anderson B E Chen I Jatoi and P S RosenbergldquoEffects of estrogen receptor expression and histopathology onannual hazard rates of death from breast cancerrdquo Breast CancerResearch and Treatment vol 100 no 1 pp 121ndash126 2006

[4] S Sangrajrang P Schmezer I Burkholder et al ldquoPolymor-phisms in three base excision repair genes and breast cancer riskinThai womenrdquo Breast Cancer Research and Treatment vol 111no 2 pp 279ndash288 2008

[5] L Gros M K Saparbaev and J Laval ldquoEnzymology of therepair of free radicals-inducedDNAdamagerdquoOncogene vol 21no 58 pp 8905ndash8925 2002

[6] G L Dianov and U Hubscher ldquoMammalian base excisionrepair the forgotten archangelrdquo Nucleic Acids Research vol 41no 6 pp 3483ndash3490 2013

[7] S Mitra T Izumi I Boldogh K K Bhakat J W Hill andT K Hazra ldquoChoreography of oxidative damage repair inmammalian genomesrdquo Free Radical Biology and Medicine vol33 no 1 pp 15ndash28 2002

[8] P W Doetsch and R P Cunningham ldquoThe enzymologyof apurinicapyrimidinic endonucleasesrdquo Mutation ResearchDNA Repair vol 236 no 2-3 pp 173ndash201 1990

[9] B Demple and L Harrison ldquoRepair of oxidative damage toDNA enzymology and biologyrdquoAnnual Review of Biochemistryvol 63 pp 915ndash948 1994

[10] G Barzilay and I D Hickson ldquoStructure and function ofapurinicapyrimidinic endonucleasesrdquo BioEssays vol 17 no 8pp 713ndash719 1995

[11] K K Bhakat A K Mantha and S Mitra ldquoTranscriptional reg-ulatory functions of mammalian AP-endonuclease (APE1Ref-1) an essential multifunctional proteinrdquo Antioxidants amp RedoxSignaling vol 11 no 3 pp 621ndash637 2009

[12] S Thakur B Sarkar R P Cholia N Gautam M Dhiman andA K Mantha ldquoAPE1Ref-1 as an emerging therapeutic targetfor various human diseases phytochemical modulation of its

Disease Markers 9

functionsrdquo Experimental and Molecular Medicine vol 46 no 7article e106 2014

[13] H W Mohrenweiser D M Wilson III and I M JonesldquoChallenges and complexities in estimating both the functionalimpact and the disease risk associated with the extensivegenetic variation in human DNA repair genesrdquo MutationResearchFundamental and Molecular Mechanisms of Mutagen-esis vol 526 no 1-2 pp 93ndash125 2003

[14] A Memisoglu and L Samson ldquoContribution of base excisionrepair nucleotide excision repair and DNA recombination toalkylation resistance of the fission yeast Schizosaccharomycespomberdquo Journal of Bacteriology vol 182 no 8 pp 2104ndash21122000

[15] B A Manvilla O Wauchope K L Seley-Radtke and A CDrohat ldquoNMR studies reveal an unexpected binding site for aredox inhibitor of AP endonuclease 1rdquo Biochemistry vol 50 no48 pp 10540ndash10549 2011

[16] L Chen C B Ambrosone J Lee T A Sellers J Pow-Sangand J Y Park ldquoAssociation between polymorphisms in theDNArepair genesXRCC1 andAPE1 and the risk of prostate cancer inwhite and black AmericansrdquoThe Journal of Urology vol 175 no1 pp 108ndash112 2006

[17] J Baute and A Depicker ldquoBase excision repair and its role inmaintaining genome stabilityrdquo Critical Reviews in Biochemistryand Molecular Biology vol 43 no 4 pp 239ndash276 2008

[18] Z Li W Guan M-X Li et al ldquoGenetic polymorphism of DNAbase-excision repair genes (APE1 OGG1 and XRCC1) and theircorrelation with risk of lung cancer in a Chinese populationrdquoArchives of Medical Research vol 42 no 3 pp 226ndash234 2011

[19] R D Mittal R K Mandal and R Gangwar ldquoBase excisionrepair pathway genes polymorphism in prostate and bladdercancer risk in North Indian populationrdquoMechanisms of Ageingand Development vol 133 no 4 pp 127ndash132 2012

[20] T Sliwinski P Ziemba Z Morawiec M Kowalski MZadrozny and J Blasiak ldquoPolymorphisms of the DNA poly-merase beta gene in breast cancerrdquo Breast Cancer Research andTreatment vol 103 no 2 pp 161ndash166 2007

[21] M S Alanazi N R Parine J P Shaik H A Alabdulkarim S AAjaj and Z Khan ldquoAssociation of single nucleotide polymor-phisms in Wnt signaling pathway genes with breast cancer inSaudi patientsrdquo PLoS ONE vol 8 no 3 Article ID e59555 2013

[22] K J Livak ldquoAllelic discrimination using fluorogenic probes andthe 51015840 nuclease assayrdquo Genetic Analysis Biomolecular Engineer-ing vol 14 no 5-6 pp 143ndash149 1999

[23] A Roy A Kucukural and Y Zhang ldquoI-TASSER a unified plat-form for automated protein structure and function predictionrdquoNature Protocols vol 5 no 4 pp 725ndash738 2010

[24] A Sali L Potterton F Yuan H Van Vlijmen and M KarplusldquoEvaluation of comparative protein modeling by MODELLERrdquoProteins Structure Function andGenetics vol 23 no 3 pp 318ndash326 1995

[25] M Wiederstein and M J Sippl ldquoProSA-web interactive webservice for the recognition of errors in three-dimensionalstructures of proteinsrdquoNucleic Acids Research vol 35 no 2 ppW407ndashW410 2007

[26] Y Dehouck J M Kwasigroch D Gilis and M RoomanldquoPoPMuSiC 21 a web server for the estimation of proteinstability changes uponmutation and sequence optimalityrdquoBMCBioinformatics vol 12 article 151 2011

[27] V Parthiban M M Gromiha and D Schomburg ldquoCUPSATprediction of protein stability upon point mutationsrdquo NucleicAcids Research vol 34 pp W239ndashW242 2006

[28] H Venselaar T A H te Beek R K P Kuipers M L Hekkel-man and G Vriend ldquoProtein structure analysis of mutationscausing inheritable diseases An e-Science approach with lifescientist friendly interfacesrdquo BMC Bioinformatics vol 11 article548 2010

[29] M Alanazi A A Pathan Z Abduljaleel et al ldquoAssociationbetween PARP-1 V762A polymorphism and breast cancersusceptibility in Saudi populationrdquo PLoS ONE vol 8 no 12Article ID e85541 2013

[30] B R Brooks C L Brooks III A D Mackerell Jr et alldquoCHARMM the biomolecular simulation programrdquo Journal ofComputational Chemistry vol 30 no 10 pp 1545ndash1614 2009

[31] M Alanazi A S Al-Arfaj Z Abduljaleel et al ldquoNovel hypox-anthine guanine phosphoribosyltransferase gene mutations inSaudi Arabian hyperuricemia patientsrdquo BioMed Research Inter-national vol 2014 Article ID 290325 12 pages 2014

[32] M Alanazi A A K Pathan S A Ajaj et al ldquoDna repair genesXRCC1 XRCC3 XPD ANDOGG1 polymorphisms among thecentral region population of Saudi Arabiardquo Biological Researchvol 46 no 2 pp 161ndash167 2013

[33] R D Wood M Mitchell J Sgouros and T Lindahl ldquoHumanDNA repair genesrdquo Science vol 291 no 5507 pp 1284ndash12892001

[34] T Izumi T K Hazra I Boldogh et al ldquoRequirement forhuman AP endonuclease 1 for repair of 3rsquo-blocking damage atDNA single-strand breaks induced by reactive oxygen speciesrdquoCarcinogenesis vol 21 no 7 pp 1329ndash1334 2000

[35] M R Kelley DNA Repair in Cancer Therapy Molecular Targetsand Clinical Applications Academic Press 2012

[36] Z Zhao C Liu Y Zeng et al ldquoThe association between theAPE1 Asp148Glu polymorphism and breast cancer suscepti-bility a meta-analysis based on case-control studiesrdquo TumorBiology vol 35 no 5 pp 4727ndash4734 2014

[37] M A F El-Hazmi A R Al-Swailem A S Warsy A M Al-Swailem R Sulaimani and A A Al-Meshari ldquoConsanguinityamong the Saudi Arabian populationrdquo Journal of MedicalGenetics vol 32 no 8 pp 623ndash626 1995

[38] M Ema K Hirota J Mimura et al ldquoMolecular mechanismsof transcription activation by HLF and HIF1alpha in responseto hypoxia their stabilization and redox signal-induced inter-action with CBPp300rdquo The EMBO Journal vol 18 no 7 pp1905ndash1914 1999

[39] C Gaiddon N C Moorthy and C Prives ldquoRef-1 regulates thetransactivation and pro-apoptotic functions of p53 in vivordquoTheEMBO Journal vol 18 no 20 pp 5609ndash5621 1999

[40] G Tell L Pellizzari D Cimarosti C Pucillo and G DamanteldquoRef-1 controls pax-8 DNA-binding activityrdquo Biochemical andBiophysical Research Communications vol 252 no 1 pp 178ndash183 1998

[41] S Xanthoudakis G Miao FWang Y-C E Pan and T CurranldquoRedox activation of Fos-Jun DNA binding activity is mediatedby a DNA repair enzymerdquoThe EMBO Journal vol 11 no 9 pp3323ndash3335 1992

[42] A Y Shaikh and L J Martin ldquoDNA base-excision repairenzyme apurinicapyrimidinic endonucleaseredox factor-1 isincreased and competent in the brain and spinal cord ofindividuals with amyotrophic lateral sclerosisrdquoNeuroMolecularMedicine vol 2 no 1 pp 47ndash60 2002

[43] S H Song E J Cho M S Park et al ldquoRedox regulatingprotein APE1Ref-1 expression is increased in abdominal aorticcoarctation-induced hypertension ratsrdquo Journal of the KoreanSociety of Hypertension vol 18 no 3 pp 126ndash135 2012

10 Disease Markers

[44] A Al-Attar L Gossage K R Fareed et al ldquoHuman apurinicapyrimidinic endonuclease (APE1) is a prognostic factor inovarian gastro-oesophageal and pancreatico-biliary cancersrdquoBritish Journal of Cancer vol 102 no 4 pp 704ndash709 2010

[45] V Di Maso C Avellini L S Croce et al ldquoSubcellular localiza-tion ofAPE1Ref-1 in humanhepatocellular carcinoma possibleprognostic significancerdquoMolecular Medicine vol 13 pp 89ndash962007

[46] G Tell F Quadrifoglio C Tiribelli and M R Kelley ldquoThemany functions of APE1Ref-1 not only a DNA repair enzymerdquoAntioxidants and Redox Signaling vol 11 no 3 pp 601ndash6192009

[47] E Canbay B Agachan M Gulluoglu et al ldquoPossible associa-tions of APE1 polymorphism with susceptibility and HOGG1polymorphism with prognosis in gastric cancerrdquo AnticancerResearch vol 30 no 4 pp 1359ndash1364 2010

[48] C Liu Q Yin L Li Y-Z Zhuang X Zu and Y WangldquoAPE1 Asp148Glu gene polymorphism and bladder cancer riska meta-analysisrdquo Molecular Biology Reports vol 40 no 1 pp171ndash176 2013

[49] M Pieretti N H Khattar and S A Smith ldquoCommon polymor-phisms and somatic mutations in human base excision repairgenes in ovarian and endometrial cancersrdquoMutationResearchmdashMutation Research Genomics vol 432 no 3-4 pp 53ndash59 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

of the damaged base apurinicapyrimidinic endonuclease 1(APE1) hydrolytically cleaves the phosphodiester backbone 51015840to the AP site resulting in the formation of a 51015840-deoxyribosephosphate (51015840-dRP) and a 31015840-OH primer [8] At this junctureDNA polymerase then inserts a correct nucleotide and DNAligases seal the repaired DNA strand APE1 is a multifunc-tional enzyme which is located on chromosome 14q112ndashq12 [9] It exhibits DNA repair activity and has a role inthe reductive activation of many transcription factors Thesetwo functions are encoded by two different sites of APE1enzyme The N-terminal region encodes the redox functionand the C-terminal region encodes the repair function [10]The DNA repair activity includes 31015840-phosphodiesterase 31015840-phosphatase and 3-51015840-exonuclease activities [11] To coordi-nate BER pathway APE1 interacts with PARP1 XRCC1 DNApolymerase 120573 and flap endonuclease 1 (FEN1) [12] APE1stimulates all these proteins individually DNA polymerasethen inserts a correct nucleotide and DNA ligases seal therepaired DNA strand Mutations in this highly regulatedmechanism can be caused by single nucleotide polymor-phisms (SNPs) which will result in insufficient DNA repairwhich may enhance DNA lesions [13 14] Mutations in APE1gene may affect its function If AP sites are not processedmutagenesis and cellular cytotoxicity can result from blockedDNA replication machinery or from misincorporation ofbases opposite theAP site [12 15]TheAPE1 polymorphism atcodon 148 has been previously reported to be associated withprostate cancer risk [14] However previous reports on theassociation between APE1 polymorphisms and cancer riskhave provided inconsistent results [16ndash19] Data pertainingthe association of the APE1 polymorphisms with breastcancer risk are also inconsistent [4 20] To the best of ourknowledge there is no published report on the associationbetweenAPE1 SNP variant Asp148Glu (rs1130409) and breastcancer in Saudi population Thus this is the first study thatinvestigates APE1 SNP Asp148Glu association with breastcancers using TaqMan assay in Saudi women Additionallyin silico analyses were performed to determine the structuraland functional consequence of Glu instead of Asp at codon148 of APE1 protein

2 Materials and Methods

21 Study Population This study was a case-control studythat included 100 Saudi female patients diagnosed with breastcancer at King Fahad Medical City Hospital Riyadh SaudiArabia along with 100 controls Controls were age-matchedand confirmed to be free of cancer and other diseases follow-ing thorough physical examinations The demographic dataof each patient and control were recorded (Table 1) Patientsand controls were enrolled under King Khalid UniversityHospital Institutional Review Board approved protocol withwritten informed consent

22 Genotyping Genomic DNA was extracted from theblood samples of breast cancer cases and controls usingQIAmp DNA blood Mini Kit (Qiagen Valencia CA) follow-ing the manufacturerrsquos instructions SNP rs1130409 in APE1gene was genotyped using TaqMan allelic discriminationassay as described previously [21 22] Ten percent of the

Table 1 Clinical characteristics of study subjects

Variable Character Number of samplesAge (years)Median age (48 plusmn 82lowast) lt48gt48 4753

Estrogen receptor ER+ERminus 5545Progesterone receptor PR+PRminus 5644HER status HER+HERminus 4057lowastMedian age with standard deviation

samples were subjected to repeated analysis for verificationof genotyping procedures

23Modeling ofMutant Structure X-ray diffraction structureof APE1 (2O3H) from PDB database was used as a referenceto compare the wild-type (Asp148) protein structure withthe predicted mutant structure (148Glu) and its solventaccessibility including secondary structures was modeledusingmolecular dynamics (MD) simulationThe best homol-ogy model for APE1 protein with Asp148Glu was selectedusing I-TASSER server [23] The mutant APE1 Asp148Glu3-D structure was predicted using Modeller 9v10 [24] Thepredicted model for mutant type protein was evaluated usingProSA-web [25]

24 Analyzing the Effects of Mutation on Protein StabilityStability of the mutant protein is checked using prediction ofPrediction of Protein Mutant Stability Changes (PoPMuSiC)[26] The results were based on the selected ΔΔ119866 values inkcalmol of the predictedAPE1Asp148Glu structure to evalu-ate the change in folding free energy after mutation (ΔΔ119866)Additionally CUPSAT (Cologne University Protein StabilityAnalysis Tool) was also used to confirm the results [27]

25 The Effect of Mutant Residue 148Glu on APE1 Structureand Function The presence of glutamate instead of aspartateat codon 148 of APE1 may affect the overall structure with apotential to alter its activityThe effect of Asp148Glumutationwas analyzed using Have yOur Protein Explained (HOPE)program [28] as described earlier by Alanazi et al [29]

26 Molecular Dynamics Simulation The effect of glutamateat codon 148 on APE1 was studied by comparing with three-dimensional structure of APE1 protein present in the proteindatabank [PDB 2O3H] Structures deduced forAPE1 harbor-ingmutationAsp148Gluwhich is identified to be risk in Saudibreast cancer samples were utilized for the analyses Biopoly-mer module in InsightII was used to substitute aspartate withglutamate residue at codon 148 from the fragment library andto addhydrogen atoms to bothwild-type andmutated proteinstructures at pH 70 (Accelrys Inc San Diego CA) Themolecular simulation programCHARMMwas used to derivethe force fields of both structures [30] A sequence of energyminimization steps were performed as described by Alanaziet al [31] on native and mutant protein structures by usingInsightIIDiscover (Accelrys Inc San Diego CA) Followingenergy minimization protein structures were analyzed usingDiscovery Studio 25 (Accelrys Inc San Diego CA)

Disease Markers 3






3 Results




4 Disease Markers







AspGlu freq(number)

GluGlu freq(number)

Wild typeAsp

VariantGlu


YRI (119899 = 226) 0133 0381 0487 032 068 924 0002lowast






Disease Markers 5

HO

O

OH

O

148

OH

O

OH

O


H2NH2N

Mutates to Overlap










6 Disease Markers


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5minus513

minus813








Disease Markers 7


Trun

cate

d oc

tahe

dron

bou

ndar

y ex

plic

it w

ater

solv

ated

syste

m 5

700

wat

er m

olec

ules

and

solv

ate

Chain A

Chain B Chain C

Chain A


prot

ein

with

edge

dist

ance

10Aring

cuto

ff

Asp148


Asp148 = Glu

Loop10

Asp148=

Glu

(a)

(b)

(c)






8 Disease Markers




Abbreviations









Acknowledgment


References













Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 3






3 Results




4 Disease Markers







AspGlu freq(number)

GluGlu freq(number)

Wild typeAsp

VariantGlu


YRI (119899 = 226) 0133 0381 0487 032 068 924 0002lowast






Disease Markers 5

HO

O

OH

O

148

OH

O

OH

O


H2NH2N

Mutates to Overlap










6 Disease Markers


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5minus513

minus813








Disease Markers 7


Trun

cate

d oc

tahe

dron

bou

ndar

y ex

plic

it w

ater

solv

ated

syste

m 5

700

wat

er m

olec

ules

and

solv

ate

Chain A

Chain B Chain C

Chain A


prot

ein

with

edge

dist

ance

10Aring

cuto

ff

Asp148


Asp148 = Glu

Loop10

Asp148=

Glu

(a)

(b)

(c)






8 Disease Markers




Abbreviations









Acknowledgment


References













Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Disease Markers







AspGlu freq(number)

GluGlu freq(number)

Wild typeAsp

VariantGlu


YRI (119899 = 226) 0133 0381 0487 032 068 924 0002lowast






Disease Markers 5

HO

O

OH

O

148

OH

O

OH

O


H2NH2N

Mutates to Overlap










6 Disease Markers


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5minus513

minus813








Disease Markers 7


Trun

cate

d oc

tahe

dron

bou

ndar

y ex

plic

it w

ater

solv

ated

syste

m 5

700

wat

er m

olec

ules

and

solv

ate

Chain A

Chain B Chain C

Chain A


prot

ein

with

edge

dist

ance

10Aring

cuto

ff

Asp148


Asp148 = Glu

Loop10

Asp148=

Glu

(a)

(b)

(c)






8 Disease Markers




Abbreviations









Acknowledgment


References













Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 5

HO

O

OH

O

148

OH

O

OH

O


H2NH2N

Mutates to Overlap










6 Disease Markers


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5minus513

minus813








Disease Markers 7


Trun

cate

d oc

tahe

dron

bou

ndar

y ex

plic

it w

ater

solv

ated

syste

m 5

700

wat

er m

olec

ules

and

solv

ate

Chain A

Chain B Chain C

Chain A


prot

ein

with

edge

dist

ance

10Aring

cuto

ff

Asp148


Asp148 = Glu

Loop10

Asp148=

Glu

(a)

(b)

(c)






8 Disease Markers




Abbreviations









Acknowledgment


References













Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Disease Markers


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5


X-rayNMR

0

5

10

Z-s

core

minus20

minus15

minus10

minus5minus513

minus813








Disease Markers 7


Trun

cate

d oc

tahe

dron

bou

ndar

y ex

plic

it w

ater

solv

ated

syste

m 5

700

wat

er m

olec

ules

and

solv

ate

Chain A

Chain B Chain C

Chain A


prot

ein

with

edge

dist

ance

10Aring

cuto

ff

Asp148


Asp148 = Glu

Loop10

Asp148=

Glu

(a)

(b)

(c)






8 Disease Markers




Abbreviations









Acknowledgment


References













Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 7


Trun

cate

d oc

tahe

dron

bou

ndar

y ex

plic

it w

ater

solv

ated

syste

m 5

700

wat

er m

olec

ules

and

solv

ate

Chain A

Chain B Chain C

Chain A


prot

ein

with

edge

dist

ance

10Aring

cuto

ff

Asp148


Asp148 = Glu

Loop10

Asp148=

Glu

(a)

(b)

(c)






8 Disease Markers




Abbreviations









Acknowledgment


References













Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 Disease Markers




Abbreviations









Acknowledgment


References













Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 9

































10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















10 Disease Markers












of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Association of DNA Repair Gene APE1...

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