Germline genomic instability in PCNA mutants of Drosophila: DNA fingerprinting and microsatellite...

Mutation Research 570 (2005) 253–265

Germline genomic instability inPCNAmutants of Drosophila:DNA fingerprinting and microsatellite analysis

Arturo Lopez, Noel Xamena, Ricard Marcos, Antonia Velazquez∗

Grup de Mutagènesi, Unitat de Genètica, Edifici Cn, Departament de Genètica i de Microbiologia,Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain

Received 6 September 2004; received in revised form 4 November 2004; accepted 26 November 2004Available online 5 January 2005

Abstract

PCNA participates in multiple processes of DNA metabolism with an essential role in DNA replication and intervening inDNA repair. Temperature-sensitive PCNA mutants of Drosophila (mus209) are sensitive to mutagens, impair developmentalprocesses and suppress positional-effect variegation. To investigate the role of proliferating cell nuclear antigen (PCNA) ingermline genomic stability, independentmus209-defective andmus209-normal lines were established and maintained over sixgenerations. A time course study was carried out and general genomic alterations were analyzed in the progeny by usingarbitrarily primed PCR (AP-PCR) and microsatellite analysis. The AP-PCR analysis has shown that a dysfunctional PCNAleads to germline genomic instability, being the amount of genomic alterations transmitted to the progeny directly related tot B1 increaseolc lls, bothi mici©

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he number ofmus209 mutant alleles. In addition, we have found that the frequency of genomic alterations tends tover successive generations. Surprisingly, the highest microsatellite instability was found in the heterozygousmus209-defective

ines, suggesting a greater mutation rate in these individuals, in comparison with the homozygousmus209-defective lines. Inonclusion, our results clearly indicate that PCNA is an important factor to maintain genomic stability in germinal cen the overall genome and in simple repeated sequences. The implication ofPCNAmutations in transgenerational genonstability and related to cancer susceptibility is also discussed.

2004 Elsevier B.V. All rights reserved.

eywords:PCNA; Genomic instability; Germline mutations; Drosophila;mus209mutant; DNA fingerprinting

∗ Corresponding author. Tel.: +34 935 813 111;ax: +34 935 812 387.

E-mail address:[email protected] (A. Velazquez).

1. Introduction

Genomic stability is guaranteed by the proper fution of DNA metabolism. Hence, alterations in mecnisms such as DNA replication, recombination, repor cell-cycle control lead to genetic changes withmatic consequences to organisms. All these proc

027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2004.11.005

254 A. Lopez et al. / Mutation Research 570 (2005) 253–265

share some common factors; thus, a lack of functionof any of these factors is expected to generate multi-ple genomic alterations in the cells. On the other hand,the mutator phenotypes associated to these deficien-cies are characterized by an increased accumulation ofDNA damage, a phenomenon well known as a puta-tive cause of cancer[1]. The proliferating cell nuclearantigen (PCNA) gene belongs to the group of genesinvolved in multiple processes of DNA metabolism forwhich a mutation suppressor effect has been reported[2,3].

PCNA is a highly conserved protein that forms a ho-motrimeric complex that encircles double-strand DNAand slides along it, serving as the platform to bind di-verse proteins[2–4]. PCNA is essential for DNA repli-cation and also participates in DNA repair. In DNAreplication, PCNA is the processivity factor of DNApolymerases[5,6] and stimulates the activity of FEN-1,involved in Okazaki-fragment maturation[7,8]. It hasalso been reported that PCNA interacts with proteinsinvolved in DNA repair[9–15] and in cell cycle con-trol [16–19]. The wide capacity of PCNA to bind suchdiverse proteins suggests the important role of this fac-tor in coordinating the cell cycle and DNA replicationprocesses with DNA repair[17,18].

Yeast PCNA mutants are sensitive to mutagens, havegrowth defects, and show an increased spontaneousmutation frequency[20,21]. Moreover, further stud-ies [22,23] on the mutator phenotype of yeast PCNAmutants suggest that many of these mutants are si-m ses,c stud-i us,i innds tchrw redD

ses[ smsh sta-b hilaP or-m lls.Da d

�-rays, indicates the participation of this gene in DNArepair[33]. Thus, genetic studies with Drosophila hadrevealed the participation of PCNA in DSB repair[32]and in development processes[34]; furthermore, itsimplication in chromatin structure has also been sug-gested[33,35]. Recently we have been shown the im-plication of PCNA to maintain microsatellite stabilityin Drosophila germ cells[36].

In the present study, we have evaluated the impli-cation of PCNA in maintaining transgenerational ge-nomic stability. To carry out this study, intrabreed linesof individuals bearing different copies of a PCNA mu-tant allele, as well as normal lines for PCNA were es-tablished. The global genomic stability in the germlinealong successive generations was estimated, by ana-lyzing the alterations in the progeny at specific timeof study and compared to the alterations of founders.The genomic analysis has been carried out by usingthe arbitrarily primed PCR (AP-PCR) technique thatallows to analyze random sequences representatives ofthe overall genome[37,38]. Previously, we have shownthat this approach is useful to quantify inherited ge-nomic damage in Drosophila[39,40], as well as to de-tect tumor genomic alterations[41]. In addition, otherauthors have also reported the use of AP-PCR to de-tect inherited DNA alterations[42–44]. The analysisof the profiles obtained by AP-PCR together with themicrosatellite analysis reported here, show that PCNAplays an important role to maintain global germlinegenomic stability in vivo.

2

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m n-d tonyB ificgm atpT ilst[m rtilea

ultaneously defective in multiple cellular procesausing more than one mutagenic defect. Otheres have also implicated PCNA in DNA repair; thn vitro assays revealed that PCNA participatesucleotide and base excision repair[24–28], and inouble-strand breaks (DSB) repair[29]. Yeast in vivotudies have shown that PCNA is involved in mismaepair (MMR) [9,30] and postreplication repair[31],hilst Drosophila PCNA mutants showed impaiSB repair.[32].Given that PCNA is essential in cellular proces

2,3], to understand its role in high eukaryote organias a great interest, mainly in relation with genomicility. To this respect, genetic analysis of DrosopCNA mutants can provide important in vivo infation about their mutator effects in germinal cerosophila PCNA is encoded by themus209gene[33]nd the sensitivity of themus209mutants to MMS an

. Materials and methods

.1. Drosophila stocks and sets of crosses

The PCNA mutant mus209B1: b pr cn bwus209B1/Cy, was kindly provided by Daryl S. Heerson (Pharmacological Science, Suny at Srook, New York). For more details on the specenetic markers, please refer to FlyBase[45]. Theus209B1 carries a proline-to-leicine substitutionosition 140, which is a highly conserved residue[34].hemus209B1mutant is a heat-sensitive lethal that fa

o complete development when it is cultured at 29◦C46]. In addition, homozygousmus209B1/mus209B1 fe-ales are unconditionally sterile, but males are fet permissive temperature, 22± 1◦C[33]. TheCanton-

A. Lopez et al. / Mutation Research 570 (2005) 253–265 255

S strain, maintained in our laboratory, was used as awild-type PCNA strain.

Three sets of crosses were established byselecting homozygousmus209B1/mus209B1, het-erozygous mus209B1/Cy, or Canton-S males asparentals of six successive generations. For set (1),mus209B1/mus209B1 males andmus209B1/Cy virginfemales were selected and mated individually tostart 25 lines. From the offspring of these crosses,mus209B1/mus209B1 sons andmus209B1/Cy virgindaughters were selected and mated to a ratio of twomales with three females. To maintain these lines,the same selection procedure was followed everygeneration. For set (2),mus209B1/Cy males andmus209B1/Cy virgin females were selected and matedindividually to constitute 27 lines. In each generation,mus209B1/Cysons and virgin daughters were selectedfrom the offspring and mated as in set (1). In the caseof set (3),Canton-Smales and virgin females weremated individually to constitute 20 lines that weremaintained as set (1) and (2). These sets of crosseswere calledmus209−/−, mus209+/− and mus209+/+

lines, respectively. The lines were maintained at23± 1◦C for six generations. The founders maleand female of each line, as well as the offspring atgeneration 3 (G3) ofmus209−/− andmus209+/+ lines,and the offspring at generation 6 (G6) ofmus209−/−,mus209+/− and mus209+/+ lines were collected andkept at−80◦C.

2

-n e-sd

2

redb -da8dp rWa her-

mal Controller (PT-100 MJ Research, USA). Apreincubation for 3 min at 94◦C was followed forfive cycles in low stringency conditions (94◦C for30 s, 40◦C for 30 s, and 72◦C for 90 s) and thenfor 30 cycles in high stringency conditions (94◦Cfor 15 s, 55◦C for 15 s, and 72◦C for 60 s). Fi-nally, 3�l of reaction was fractionated by elec-trophoresis on a 6% denaturing polyacrylamidegel (6 h at 45 W) and visualized by autoradiogra-phy.

2.4. Quantification of genomic damage byAP-PCR

Fingerprinted bands obtained by AP-PCR dependon the primer used and the genome to be analyzed.Assessment of genomic damage by this technique re-quires to identify those reproducible and clearly visu-alized bands in the autoradiography of each repeatedexperiment. As in our previous studies[39–41], onlythe reproducible bands were considered in the analysis.To estimate the total genetic alterations accumulated atthe different studied times, G3 and G6 inmus209−/−and inmus209+/+ lines, and G6 inmus209+/− lines,the number of altered bands in the progeny of eachline were scored. The fingerprint alterations consid-ered in this study were new bands and mobility shiftsof bands; loss of bands were not score since the dis-appearance of bands in the progeny fingerprint couldbe due to the transmission of AP-PCR untraceable al-l ocusb anda hus,a newb fileso otaln na-l ithp -s te oft tiono F)w e wasu ual,i eent t byt xont

.2. Genomic DNA isolation

Individual flies kept at−80◦C were used for geomic DNA extractions, following the method dcribed by Roberts[47] and dissolved in 20–30�l ofouble-distilled water.

.3. AP-PCR amplification

Twenty-five microliters of reaction was prepay using 1�l of the DNA stock solution from inividual flies (∼50 ng of DNA) in 1× PCR re-ction buffer (50 mM KCl, 10 mM Tris–HCl pH.4, 0.01% gelatin), 4 mM MgCl2, 125�M of eachNTP, 3�Ci of [�-33P]dATP, 1 U of Taq DNAolymerase and 0.25�M of the arbitrary primeB (5′-GTTAGGGAGCCGATAAAGAG-3′). The re-

ctions were carried out in a Programmable T

eles when the founders are heterozygous for a learing a detectable allele (fingerprinted band)undetectable allele (non-fingerprinted band). T

t each point of study, the sum of the changes (ands and band shifts) found in the displayed prof the total analyzed progeny, was divided by the tumber of reproducible amplified bands (no. of a

yzed individuals× 28 or 27 fingerprinted bands wrimer WB inmus209andCanton-Sindividuals, repectively). This fraction was used as an estimahe total damage (damage fraction, DF). The fracf mobility shifts of bands (band shifts fraction, BSas also calculated separately. The same procedursed to estimate the damage per line or per individ

n the course time study. Statistical analysis betwhe different lines and point of study was carried ouhe Fisher’s exact test, two-tailed, and the Wilconest.


2.5. Microsatellite analysis

The microsatellite loci, Dronanos, Droabdb,Dmsgg3, Mam and DmtenA, described by Schuget al. [48] were analysed by locus specific PCR.The primers to amplify the microsatellite loci, aswell as the PCR conditions used in this studyhave been previously described by us[39,40]. PCRproducts were separated on 6% denaturing poly-acrylamide gels and visualized by autoradiogra-phy. The Fisher’s exact test, two-tailed, and theWilconxon test were used for statistical analy-sis.

3. Results

3.1. Genomic DNA profiles reveal geneticvariation in the mus209B1 strain

As expected, the displayed AP-PCR DNA profilesof mus209B1 flies are different from those obtainedof normal flies (Canton-Sstrain), due to polymor-phic differences between these strains (seeFig. 1).All our previous AP-PCR experiments carried outwith the Canton-Sstock have shown no differencesin the genomic profiles ofCanton-Sindividuals([36],and unpublished results). However, after analyzing 60

Fm

ig. 1. AP-PCR profiles of genomic DNA of homozygousmus209B1/musus209B1/mus209B1 individuals is indicated with an arrow. Sizes of the

209B1 andCanton-Sindividuals. Example of differences betweenbands are shown on the right.


homozygousmus209B1/mus209B1 flies, 14 out of 28AP-PCR fingerprinted reproducible bands were foundto be polymorphic (Fig. 1). This indicates genetic dif-ferences between individuals of themus209B1 stock,suggesting that an intrinsic mechanism subsist for ge-netic variation in this stock.

The high genetic variation observed within themus209B1 strain by AP-PCR profiles, led us to hy-pothesize that the altered PCNA in this strain must beresponsible for the high level of spontaneous mutationfound in themus209B1flies. Thus, to analyze the overallgenetic alterations taking place along successive gen-erations, a time course study was performed by startingindividual crosses of defective PCNA flies (mus209B1)and crosses of normal PCNA flies (Canton-S).

3.2. Genetic alterations detected by AP-PCR inthe mus209B1 strain

To test the genomic instability in the germlineof the PCNA mutantmus209B1, genetic alterationsin the parental germ cells transmitted to the off-spring were analyzed over several generations. Tocarry out this study two sets ofmus209B1 individ-ual crosses, calledmus209−/− andmus209+/− lines,were established and maintained for six generations.A set of Canton-Scrosses (mus209+/+ lines) wasused as a control. Themus209B1 lines were main-tained by selecting homozygousmus209B1/mus209B1

a ofe nes,m tal.S sn (seeS al-l er-e NA( oP ha mici ndi

m -n par-i Ap ate

the total damage (DF), new bands and mobility shifts ofbands were considered (see Section2). We also calcu-lated the fraction of mobility shifts separately (BSF),since this type of alteration is assumed to reflect in-stability of single repeated sequences (see bellow). InFig. 2, an example of changes detected in the DNA pro-files of the progeny of amus209−/− line at G3 and G6is shown.

We found that the number of altered bands per in-dividual depends on the parental genetic background,being in the progeny of control crosses where thelowest number of alterations was found. Thus, asshown inTable 1, at G6 the average number of alteredbands per individual was 1.65± 1.92, 0.81± 1.26 and0.03± 0.17 in the progeny ofmus209−/−, mus209+/−andmus209+/+ lines, respectively. In themus209−/−lines an increase in the number of alterations per indi-vidual from G3 to G6 was also observed (seeTable 1),indicating that in the germline of deficient PCNA flies,genetic changes emerge progressively over time.

It is important to point out that, in the analysis of theprogeny AP-PCR profiles of each line, at the points ofstudy, we did not find two or more individuals showingthe same type and number of alterations. This observa-tion indicates that, at each point of study, the progenyof any given line (family) had an independent origin;therefore, in our analysis we discard the possibility ofoverestimating the number of alterations found in thegerm cells, when some of the siblings came from thesame premeiotic event.

3o

ngg cal-c enyo r ofms thanot form -t ntlyll p-r unto ental

nd heterozygousmus209B1/Cymales as parentalsach generation, respectively. In both sets of lius209B1/Cy fertile females were used as parenincemus209B1/mus209B1 females are sterile, it waot possible to perform true homozygous linesection2). Thus, the present experimental design

owed to estimate the total genomic instability inhnt to each genetic background, defective to PCmus209−/− and mus209+/− lines), and normal tCNA (mus209+ /+ lines). In addition, this approaclso allowed to reveal possible differences in geno

nstability between families (individual crosses) anto the progeny of each set of lines.

At different times (G3 and G6 formus209−/− andus209+/+ lines and G6 formus209+/− lines), the geetic changes in each line were quantified by com

ng the DNA profiles of each emerged fly with the DNrofiles of the founders male and female. To estim

.3. Intergenerational genomic stability dependsn PCNA function

To estimate the overall genomic instability aloenerations, the fraction of genetic alterations wasulated at different times of study, for the total progf each set of crosses bearing different numbeus209mutant alleles. As shown inTable 1, after

ix intrabreeding generations, the DF was morene order of magnitude higher in themus209B1 lines

han in the control lines (0.061, 0.029 and 0.001,us209−/−, mus209+/− andmus209+/+ lines, respec

ively); being at G6 the genomic damage significaower in themus209+/− lines than in themus209−/−ines (P< 10−5). The DF of each analyzed line is reesented inFig. 3 and clearly shows that the amof genomic damage depends on the number of par


Fig. 2. Example of genomic alterations found at G6 and G3 inmus209−/−crosses: line 31. AP-PCR profiles of genomic DNA of founders male(M) and female (F) and progeny at G3 and G6 are presented. Lines on the left indicate the reproducible amplified bands used to quantify thegenomic damage. Changes in the fingerprinted bands are indicated with a black dot (•). Sizes of the bands are shown on the right.

mus209mutant alleles. In addition, in themus209−/−crosses we have observed 27% more alterations at G6than at G3 (DF at G6: 0.061, at G3: 0.048) suggest-ing a continued formation of genetic alterations overgenerations. The DF increase from G3 to G6 in themus209−/− background was also evident when the ge-netic damage in the individual lines (families) was an-alyzed separately (seeFig. 3). In this case, six out often analyzedmus209−/− lines showed a higher DF atG6 than at G3; the increase of damage being more thantwo-fold in three of the lines (lines 9, 12, 32). It is

important to consider that the lack of increase in theDF with generations observed in the lines 5, 18, 27and 35, might be explained by negative selection ofthe highly damaged individuals arising over genera-tions in these lines. This assumption would reinforcethe observed increase of genetic alterations over gener-ations, since this fact could account for the lack of sig-nificant difference found in themus209−/− lines DF,at G6 respect to G3. It is also conceivable that moregenerations could be needed to achieve a significantincrease in the number of alterations. Therefore, our


Table 1Genomic damage over time detected by AP-PCR in the progeny ofmus209−/−, mus209+/− andmus209+/+ lines

Type oflines

No. ofanalysed lines

No. of analysedindividuals

No. of analysedbands

No. of alteredbands

Mean alterations perindividual± S.D.

DFa No. of bandshifts

BSFb

mus209−/−G3 10 86 2408 116 1.31±1.34 0.048c 10 0.0040c

G6 10 92 2576 158 1.65±1.92 0.061d,ns 18 0.0070e

mus209+/−G6 9 81 2268 66 0.81±1.26 0.029e 11 0.0050e

mus209+/+

G3 10 80 2160 11 0.11±0.32 0.005 1 0.0005G6 10 99 2673 3 0.03±0.17 0.001 0 0.0000

a DF: damage fraction calculated as the total number of altered bands divided by the total number of analyzed bands (see Section2).b BSF: band shifts fraction, calculated as the total number of band shifts divided by the total number of analyzed bands.c Statistically significant respect tomus209+/+ at G3 (DF,P< 10−5; BSF,P= 0.01; Fisher’s exact test, two-tailed).d Statistically significant respect tomus209+/− andmus209+/+ lines at G6 (P< 10−5, Fisher’s exact test, two-tailed).e Statistically significant respect tomus209+/+ at G6 (DF,P< 10−5; BSF,P< 10−5 in mus209−/− andP< 10−3 in mus209+/−; Fisher’s exact

test, two-tailed).ns No statistically significant respect tomus209−/− at G3 (Wilcoxon test).

time course study put into manifest that flies with dys-functional PCNA present germline genomic instability,increasing the overall genomic alterations over succes-sive generations. Accordantly, control lines showed thelowest genomic damage, which was similar at G3 andG6. Moreover, the DF estimated in control lines wascomparable to the DF reported by us in previous controlcrosses[40].

3.4. Distribution of genetic alterations in theoffspring

To point out the variability of genomic damage intothe progeny, in relation to both the genetic backgroundand time of study, inFig. 4 the distribution of thenumber of alterations per individual is represented. Aprogressive increase in the number of alterations perindividual with the number of parentalmus209mu-tant alleles can be observed, with the progeny of themus209−/− lines at G6 showing the highest number ofalterations. Thus, in these lines 7.6% of the analyzedindividuals at G6 presented six or more alterations,with one individual showing 14 altered bands (data notshown). In themus209+/− lines at G6, only two indi-viduals (2.5%) showed a maximum of six alterations.By contrast, the number of alterations found in theprogeny of themus209+/+ lines was very low, since atG3 and at G6 only individuals with one alteration werefound.

The number of alterations per individual also in-creased over generations. Thus, in themus209−/− linesat G3, only 2.3% of individuals presented six or morealterations, compared to the 7.6% found at G6. In ad-dition, at G3 a maximum of 8 alterations were found inone individual. In themus209−/− lines the frequencyof individuals showing at least one change in the finger-printed bands at G3 and G6 (65.1 and 71.7%, respec-tively), also indicates accumulation of genomic dam-age in dysfunctional PCNA conditions. In comparison,the frequency of individuals showing alterations in themus209+/− background was 47%, and this frequencydecreased to less than 10% in themus209+/+ lines.

3.5. Germline microsatellite instability in themus209B1 strain

Since a relationship between changes in band mo-bility observed by AP-PCR and microsatellite instabil-ity has been reported[38,40,41,49,55], the fraction ofband shifts, in the AP-PCR profiles, were estimated.As shown inTable 1, at G6 the BSF of themus209+/+

lines was 0.0005, whereas a BSF of 0.007 and 0.005was found inmus209−/− andmus209+/− lines, respec-tively (P< 10−5 andP< 10−3 respect to control, re-spectively). These results suggest the implication ofPCNA in microsatellite instability.

To test the microsatellite instability in the differ-ent sets of lines, a concurrent specific PCR analysis


Fig. 3. Genomic damage fraction (DF) in the germ line, over suc-cessive generations. (A) DF inmus209−/− lines at G3 and G6, (B)DF in mus209+/− lines at G6 and (C) DF inmus209+/+ lines at G3and G6.

of five microsatellite loci, using the same DNA sam-ples than in the AP-PCR studies was carried out. Asshown in Table 2, at G6 themus209+/− lines pre-sented mutations in four out of five analyzed loci, be-ing the set of crosses with the highest mutation fre-quency (3.4× 10−2). However, at G6 themus209−/−lines showed mutation only in one or two microsatellite(overall frequency 8.5× 10−3), and no mutations werefound in themus209+/+ lines; being the mutation fre-

quency ofmus209+/− crosses statistically significantwith respect to both themus209+/+ andmus209−/−crosses (P< 10−4 andP< 10−2, respectively). No sig-nificant differences were found in the mutation fre-quency between themus209−/− andmus209+/+ crosses(P= 0.128). These results indicate that alterations onPCNA in heterozygous conditions let to germline MSI.We have reported this fact recently[36], where MSI wasanalyzed in the first generation ofmus209B1 crosses.It is important to remark that the MSI frequency foundin the germ cells ofmus209+/− lines was in the sameorder of magnitude that the MSI frequency found in thegerm cells ofMsh2knockout flies[36,50,51].

Contrary to the genomic instability results obtainedby AP-PCR, no increase of the frequency of mi-crosatellite mutations over generations was found inmus209−/− lines. Thus, in themus209−/− crosses, theMSI frequency in the G3 progeny was similar to theMSI found at G6 (9.6× 10−3 and 7.4× 10−3, respec-tively, P= 0.139; Wilconxon test). Accordantly, in themus209+/− crosses, the 3.4× 10−2 MSI frequency inthe G6 progeny was similar to the MSI frequency in theprogeny of first generation (1.8× 10−2), as previouslyreported by us[36] (P= 0.378).

4. Discussion

Drosophila PCNA mutants provide a good opportu-n aseo ighe antso li-c ane e ofc neticd muta-to sibler mics omeo is weh s toe ener-a herwt

ity to carry out complex genetic analyses to increur understanding on the function of PCNA in a hukaryote system. Previous studies of PCNA mutf D. melanogaster[32,33] demonstrated the impation of PCNA in several cellular processes, withssential role in development, in the maintenanchromatin structure, and to overcome induced geamage. Studies with yeast have also suggested a

or suppressor effect of PCNA (reviewed in[2]). Basedn these evidences, it is feasible to presume a posole of PCNA to maintain transgenerational genotability, hence guarantee the integrity of the genver successive generations. To test this hypothesave carried out a time-course study that allowed uvaluate the genomic damage arisen along the gtions in the progeny of two Drosophila strain, eitith a mutation in thePCNAgene (mus209B1) or with

he normalPCNAgene (Canton-S).


Fig. 4. Distribution of the number of alterations per individual inmus209−/−, mus209+/− andmus209+/+ lines, over successive generations.

The suitability of the AP-PCR technique to detectgenomic alterations has been reported, both in humansand experimental organisms[39–44,49,52–54]. Thegenomic differences between the compared genomeswould be observed as changes in the fingerprintedbands; thus, in Drosophila, we have applied success-fully the AP-PCR technique to detect spontaneousand induced germline genetic damage related to DNArepair deficiencies[39,40]. At the beginning of thepresent study, our first insight indicating that theDrosophilamus209B1 mutant could have an intrinsicmutator phenotype came from the observation of fre-quent differences in the DNA profiles displayed by AP-PCR between individuals of this stock. By contrast,

DNA profiles of control individuals were clearly moreuniform (seeFig. 1).

In this study, the quantification of new bands andband shifts in the AP-PCR fingerprints was reliable toestimate the genomic alterations arising in the estab-lished sets of lines. The new bands observed in the AP-PCR DNA profiles have theirs origin in point mutationstaking place in the primer annealing sequences, as wellas in genomic rearrangements[39,41]. Band mobilityin the AP-PCR fingerprints has previously been relatedto microsatellite instability[38,40,41,49,55]. Thus, thequantification of new bands and band shifts in the DNAprofiles displayed by AP-PCR revealed that a dysfunc-tional PCNA generates overall genomic instability in

Table 2Instability in microsatellite loci

Locus mus209−/− lines mus209+/− lines mus209+/+ lines

G3 G6 G6 G6

MSIa Percentageof changes




DmtenA(AT)14 2/107 1.8 0/108 0.0 5/82 6.1b 0/80 0.0Droabdb(AC)19 0/107 0.0 4/110 3.6 3/85 3.5 0/100 0.0Dmsgg3(CAG)11 0/104 0.0 0/106 0.0 3/75 4.0 0/89 0.0Mam(CAG)8 0/96 0.0 0/108 0.0 0/83 0.0 0/89 0.0Dronanos(AT)18 3/108 2.8 0/110 0.0 3/89 3.4 0/110 0.0

a MSI: total no. of changes/no. of analyzed individuals.b Significant difference againstmus209+/+ andmus209−/− lines at G6 (P< 0.05, Fisher’s exact test, two-tailed).


germ cells. This observation was reinforced by the cor-relation found between the number ofPCNAmutantalleles and the frequency of alterations in germ cells.However, given the different genetic background of thewild type (Canton-S) andmus209B1 strains, we can-not discard that other genetic factors present in themus209B1 lines could partially contribute to the geneticalterations observed in these lines. Moreover, severalfeatures emerged from the AP-PCR data in our time-course study of PCNA deficient lines, indicating an in-crease of genomic damage with successive generations:(i) in the progeny ofmus209−/− lines, an increase ofDF from G3 to G6 was observed, (ii) the increase of DFfrom G3 to G6 was observed in six out ten analyzedmus209−/− lines and (iii) the progeny ofmus209−/−lines showed major number of alterations per individ-ual in G6 than in G3. Altogether, these observationsprovide the first experimental evidence of a germlinemutator phenotype in PCNA mutants and reveal theimportance of PCNA to maintain the transgenerationalgenomic stability. These findings could also have im-plications to cancer susceptibility since PCNA variantalleles might exist in the human population, and in-dividuals with these alleles might transmit genomicalterations to their descendants. This assumption canbe extensive to somatic cell genomic stability in vivo.AP-PCR approach is not suitable to detect genomic al-teration on somatic cells, when all organism DNA isused for the analysis, but an increase of the mutationrate has been reported in yeast PCNA mutants[22,56].

d inb dsi rtedb wb ger-p f ge-n litya ourp A top ber ndb B-r rted[ airhp ta-b NAt uld

mimic FEN-1 gene mutations. In this sense, accumula-tion of DSBs and an increase in the number of deletionsand duplications, have been reported in yeast FEN-1mutants[58].

In addition to the implication of PCNA in general ge-nomic instability, the concurrent MSI analyses revealedthat PCNA is also required to maintain the stabilityof single repeated sequences in vivo. In our AP-PCRanalysis, PCNA deficient lines show a significant in-crease of BSF suggesting a role of PCNA in microsatel-lite instability. The relationship between band shiftshas previously been reported by us[40,41] and oth-ers[38,49,55]. However, we have found a lack of cor-relation between BSF and frequency of microsatellitemutations (i.e., inmus209+/− lines BSF and microsatel-lite mutations were 5× 10−3 and 3.8× 10−2, respec-tively). This discrepancy could be explained given thatthe locus-specific PCR microsatelllite analysis is moresensitive to study microsatellite instability than the AP-PCR technique based in random screening of genomicsequences.

Microsatellite instability is a phenotype used as anindicator of defective DNA mismatch repair. Recentin vitro and in vivo studies indicate that PCNA is re-quired for DNA mismatch repair[9,11,13,14,59], witha putative role in strand discrimination[9]. Moreover, adeficiency on DNA replication by dysfunctional DNApolymeraseδ, FEN-1 or PCNA is also expected to gen-erate instability of simple repetitive sequences([23],and herein references). Therefore, all these evidencesi tel-l e-p rolei nto seto aless ncest les.A thisf rtedr ites e-fi y ofm h re-po ant dt

We found than more than 80% of the DF estimateoth sets ofmus209B1 lines corresponds to new ban

n the AP-PCR DNA profiles. Previous results repoy us and other authors[39,40,57]suggest that neands changes detected by PCR-based DNA finrinting techniques are consequence of a type oomic instability that is independent of the instabirising from mismatch repair defects. Therefore,resent results indicate the important role of PCNrevent this type of genomic instability, which mayelated to the implication of PCNA in double-strareak (DSB) repair. To this respect, an impair DSepair of Drosophila PCNA mutants has been repo32] and, in addition, the role of PCNA in DSB repas already been demonstrated in yeast[29]. Anotherlausible explanation for this type of genomic insility could be related to the need of functional PC

o activate FEN-1; thus, a dysfunctional PCNA co

ndicate that PCNA mutants could manifest microsaite instability, either by impairing DNA mismatch rair or by an independent mechanism related to its

n genomic DNA replication. It is important to poiut that, under our experimental conditions, thef crosses with heterozygous deficient PCNA mhowed more alterations in microsatellite sequehan crosses with homozygous deficient PCNA malthough we do not have a feasible explanation to

act, this observation reinforces our previously repoesults[36]. The mutation frequency in microsatellequences of 3.4× 10−2 found in the heterozygous dcient PCNA crosses was similar to the frequencicrosatellite reported by us and others in mismatcair deficient conditions in Drosophila[36,50,51]andther organisms[60,61], and much more higher th

he frequency of 6× 10−6 reported in Drosophila wilype[62,63].


The few studies about genomic instability in PCNAmutants have all been performed in yeast[22,23]. Herewe have shown results using a more complex organism,such as Drosophila. Our results indicate that alterationsof PCNA function lead to different types of genomic al-terations in the progeny of affected individuals. Due tothe multifunctional properties of PCNA, it is feasible tothink that multiple cellular processes could be affectedin PCNA mutants, showing more than one mutageniceffect[22,23]. The transmission of germinal cell alter-ations to the progeny of individuals with dysfunctionalPCNA could be an important factor to cancer suscep-tibility. To this respect, particular attention should betaking to the high MSI found in heterozygous deficientPCNA conditions, since a high mutation rate in indi-viduals bearing one mutated copy of thePCNAgenemight be expected. Therefore, some effort should beaddress to find possiblePCNA variant alleles in thehuman population.

Acknowledgments

We thank Daryl Henderson for providing themus209B1 stock used in this study and Anna Peran fortechnical assistance. This work has been supported bythe Spanish Ministry of Education and Culture (PM99-0067, DGICYT; BOS2000-0329, DGICYT) and Gen-eralitat de Catalunya (SGR2002-00197). During thisw heS

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[7] X. Li, J. Li, J. Harrington, M.R. Lieber, P.M. Burgers, Laggingstrand DNA synthesis at the eukaryotic replication fork involvesbinding and stimulation of FEN-1 by proliferating cell nuclearantigen, J. Biol. Chem. 270 (1995) 22109–22112.

[8] X.T. Wu, J. Li, X.Y. Li, C.L. Hsieh, P.M.J. Burgers, M.R.Lieber, Processing of branched DNA intermediates by a com-plex of human FEN-1 and PCNA, Nucleic Acids Res. 24 (1996)2036–2043.

[9] A. Umar, A.B. Buermeyer, J.A. Simon, D.C. Thomas, A.B.Clark, R.M. Liskay, T.A. Kunkel, Requirement for PCNA inDNA mismatch repair at a step preceding DNA resynthesis,Cell 87 (1996) 65–73.

[10] R. Gary, D.L. Ludwig, H.L. Cornelius, M.A. Mcinnes, M.S.Park, The DNA repair endonuclease XPG binds to proliferat-ing cell nuclear antigen (PCNA) and shares sequence elementswith the PCNA-binding regions of FEN-1 and cyclin-dependentkinase inhibitor p21, J. Biol. Chem. 272 (1997) 24522–24529.

[11] L. Gu, Y. Hong, S. Mcculloch, H. Watanabe, G.M. Li, ATP-dependent interaction of human mismatch repair proteins and adual role of PCNA in mismatch repair, Nucleic Acids Res. 26(1998) 1173–1178.

[12] Z.O. Jonsson, R. Hindges, U. Hubscher, Regulation of DNAreplication and repair proteins through interaction with the frontside of proliferating cell nuclear antigen, EMBO J. 17 (1998)2412–2425.

[13] A.B. Clark, F. Valle, K. Drotschmann, R.K. Gary, T.A.Kunkel, Functional interaction of PCNA with MSH2–MSH6and MSH2–MSH3 complexes, J. Biol. Chem. 275 (2000)35669–35672.

[14] H. Flores-Rozas, D. Clark, R.D. Kolodner, Proliferating cellnuclear antigen and Msh2p-Msh6p interact to form an activemispair recognition complex, Nat. Genet. 26 (2000) 375–378.

[15] I. Unk, L. Haracska, X.V. Gomes, P.M. Burgers, L. Prakash,S. Prakash, Stimulation of 3′→5′ exonuclease and 3′-phosphodiesterase activities of yeast apn2 by proliferating cell

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ork, A. Lopez held a predoctoral fellowship from tpanish Ministry of Education and Culture.

eferences

[1] B. Vogelstein, K.W. Kinzler, The multistep nature of cancTrends Genet. 9 (1993) 138–141.

[2] E. Warbrick, The puzzle of PCNA’s many partners, BioEss22 (2000) 997–1006.

[3] G. Maga, U. Hubscher, Proliferating cell nuclear anti(PCNA): a dancer with many partners, J. Cell Sci. 116 (23051–3060.

[4] Z. Kelman, J. Hurwitz, Protein-PCNA interactions: a DNscanning mechanism? Trends Biochem. Sci. 23 (1236–238.

[5] T.S. Krishna, R.X-P. Kong, S. Gary, P.M. Burgers, J. KuriyCrystal structure of the eucaryotic DNA polymerase proceity factor PCNA, Cell 79 (1994) 1233–1243.

[6] T. Tsurimoto, PCNA, a multifunctional ring on DNA, BiochimBiophys. Acta 1443 (1998) 23–39.

nuclear antigen, Mol. Cell Biol. 22 (2002) 6480–6486.16] S. Waga, G.J. Hannon, P. Beach, B. Stillman, The p21 inhi

of cyclin-dependent kinases controls DNA replication by inaction with PCNA, Nature 369 (1994) 574–578.

17] H. Flores-Rozas, Z. Kelman, F.B. Dean, Z.Q. Pan, J.W. HaS.J. Elledge, M. O’Donell, J. Hurwitz, Cdk-interacting prot1 directly binds with proliferating cell nuclear antigen andhibits DNA replication catalyzed by the DNA polymerasδholoenzyme, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 868659.

18] M.L. Smith, I.T. Chen, Q. Zan, I. Bae, C.Y. Chen, T.M. GilmM.B. Kastan, P.M. OıConnor, A.J. Fornace Jr., Interaction ofp53-regulated protein Gadd45 with proliferating cell nucantigen, Science 266 (1994) 1376–1380.

19] E. Warbrick, W. Heatherington, D.P. Lane, D.M. Glover, PCbinding proteins in Drosophila melanogaster: the analysisconserved PCNA binding domain, Nucleic Acids Res. 26 (13925–3932.

20] R. Ayyagari, K.J. Implizzeri, B.L. Yoder, S.L. Gary, P.MBurgers, A mutational analysis of the yeast proliferatingnuclear antigen indicates distinct roles in DNA replicationDNA repair, Mol. Cell. Biol. 15 (1995) 4420–4429.


[21] N.S. Amin, C. Holm, In vivo analysis reveals that the interdo-main region of the yeast proliferating cell nuclear antigen isimportant for DNA replication and DNA repair, Genetics 144(1996) 479–493.

[22] C. Chen, B.J. Merrill, P.J. Lau, C. Holm, R.D. Kolodner, Sac-charomyces cerevisiae pol30 (proliferating cell nuclear antigen)mutations impair replication fidelity and mismatch repair, Mol.Cell. Biol. 19 (1999) 7801–7815.

[23] R.J. Kokoska, L. Stefanovi, A.B. Buermeyer, R.M. Kislay, T.D.Petes, A mutation of the yeast gene encoding PCNA destabilizesboth microsatellite and minisatellite DNA sequences, Genetics151 (1999) 511–519.

[24] A.F. Nichols, A. Sancar, Purification of PCNA as a nu-cleotide excision repair protein, Nucleic Acids Res. 20 (1992)2441–2446.

[25] M.K.K. Shivji, M.K. Kenny, R.D. Wood, Proliferating cell nu-clear antigen is required for DNA excision repair, Cell 69 (1992)367–374.

[26] Y. Matsumoto, K. Dim, D.F. Bogenhagen, Proliferating cell nu-clear antigen-dependent abasic site repair in Xenopus laevisoocytes: an alternative pathway of base excision DNA repair,Mol. Cell. Biol. 14 (1994) 6187–6197.

[27] G. Frosina, P. Fortini, O. Rossi, F. Carrozzino, G. Raspaglio,L.S. Cox, D.P. Lane, A. Abbondandolo, E. Dogliotti, Two path-ways for base excision repair in mammalian cells, J. Biol. Chem.271 (1996) 9573–9578.

[28] A. Klungland, T. Lindahl, Second pathway for completion ofhuman DNA base excision-repair: reconstruction with purifiedproteins and requirement for DNAase IV (FEN1), EMBO J. 16(1997) 3341–3348.

[29] A.M. Holmes, J.E. Haber, Double-strand break repair in yeastrequires both leading and lagging strand DNA polymerases,Cell 96 (1999) 415–424.

[30] R.E. Johnson, G.K. Kovvali, S.N. Guzder, N.S. Amin, C. Holm,Y. Habraken, P. Sung, L. Prakash, S. Prakash, Evidence for

NA.

[ h, L.n inad.

[ n re-NA

age-

[ u-tionding

[ ver,re-ster,

[ g,omb-erat-394.

[36] A. Baida, A. Lopez, R. Marcos, A. Velazquez, Germline mu-tations at microsatellite loci in homozygous and heterozygousmutants for mismatch repair and PCNA genes in Drosophila,DNA Repair 2 (2003) 827–833.

[37] J. Welsh, M. McClelland, Fingerprinting genomes using PCRwith arbitrary primers, Nucleic Acids Res. (1990) 7213–7218.

[38] J. Welsh, M. McClelland, Fingerprinting using arbitrarilyprimed PCR: application to genetic mapping, population bi-ology, epidemiology and detection of differentially expressedRNAs, in: K.B. Mullis, F. Ferre, R.A. Gibbs (Eds.), The Poly-merase Chain Reaction, Birkhauser, Boston, 1994, pp. 295–304.

[39] A. Lopez, N. Xamena, O. Cabre, A. Creus, R. Marcos, A.Velazquez, Analysis of genomic damage in the mutagen-sensitive mus-201 mutant of Drosophila melanogaster by arbi-trarily primed PCR (AP-PCR) fingerprinting, Mutat. Res. 435(1999) 63–75.

[40] A. Lopez, A. Baida, R. Marcos, N. Xamena, A. Velazquez,Spontaneous and bleomycin-induced genomic alterations in theprogeny of Drosophila treated males depends on the Msh2status. DNA fingerprinting analysis, DNA Repair 1 (2002)941–954.

[41] M.A. Peinado, S. Malkhosyan, A. Velazquez, M. Perucho, Isola-tion and characterization of allelic losses and gains in colorectaltumors by arbitrarily primed polymerase chain reaction, Proc.Natl. Acad. Sci. U.S.A. 89 (1992) 10065–10069.

[42] Y. Kubota, A. Shimada, A. Shima, DNA alterations detectedin the progeny of paternally irradiated Japanese medaka fish(Oryzias latipes), Proc. Natl. Acad. Sci. U.S.A. 92 (1995)330–334.

[43] A. Shimada, A. Shima, Combination of genomic DNA-fingerprinting into the medaka specific-locus test system forstudying environmental germ-line mutagenesis, Mutat. Res.399 (1998) 149–165.

[44] G. Vasil’eva, V.G. Bezlepkin, M.F. Lomaeva, N.P. Sirota, A.I.y of5

[ nome30

na.

[ o-gen-Res.

[ od,IRL

[ .M.mi-98)

[ Pe-se-

nesis,

involvement of yeast proliferating cell nuclear antigen in Dmismatch repair, J. Biol. Chem. 271 (1996) 27987–27990

31] C.A. Torres-Ramos, B.L. Yoder, P.M.J. Burgers, S. PrakasPrakash, Requirement of proliferating cell nuclear antigeRAD-dependent postreplication DNA repair, Proc. Natl. AcSci. U.S.A. 93 (1996) 9676–9681.

32] D.S. Henderson, D.M. Glover, Chromosome fragmentatiosulting from an inability to repair transposase-induced Ddouble-strand breaks in PCNA mutants of Drosophila, Mutnesis 13 (1998) 57–60.

33] D.S. Henderson, S.S. Banga, T.A. Grigliatti, J.B. Boyd, Mtagen sensitivity and suppresion of position-effect variegaresult from mutations in mus209, the Drosophila gene encoPCNA, EMBO J. 13 (1994) 1450–1459.

34] D.S. Henderson, U.K. Wiegand, D.G. Norman, D.M. GloMutual correction of faulty PCNA subunits in temperatusensitive lethal mus209 mutants of Drosophila melanogaGenetics 154 (2000) 1721–1733.

35] Y. Yamamoto, F. Girard, B. Bello, M. Affolter, W.J. GehrinThe cramped gene of Drosophila is a member of the Polycgroup, and interacts with mus209, the gene encoding prolifing cell nuclear antigen, Development 124 (1997) 3385–3

Gaziev, AP-PCR assay of DNA alterations in the progenmale mice exposed to low-level�-radiation, Mutat. Res. 48(2001) 133–141.

45] Flybase, The Flybase database of the Drosophila GeProjects and community literature, Nucleic Acids Res.(2002) 106–108 (available from http://flybase.bio.indiaedu/).

46] D.S. Henderson, D.A. Bailey, D.A. Sinclair, T.A. Grigliatti, Islation and characterization of second chromosome mutasensitive mutations in Drosophila melanogaster, Mutat.177 (1987) 83–93.

47] D.B. Roberts, Preparation of nucleic acids, in: D. RickwoB.D. Hames (Eds.), Drosophila, A Practical Approach,Press, Oxford, 1986, pp. 278–279.

48] M.D. Schug, K.A. Witterstrand, M.S. Gaudette, R.H. Lim, CHutter, C.F. Aquadro, The distribution and frequency ofcrosatellite loci in Drosophila melanogaster, Mol. Ecol. 7 (1957–70.

49] Y. Ionov, M.A. Peinado, S. Malkhosyan, D. Shibata, M.rucho, Ubiquitous somatic mutations in simple repeatedquences reveal a new mechanism for colonic carcinogeNature 363 (1993) 558–561.


[50] A. Lopez, N. Xamena, R. Marcos, A. Velazquez, Germ cellsmicrosatellite instability. The effect of different mutagens in amismatch repair mutant of Drosophila (spel1), Mutat. Res. 514(2002) 87–94.

[51] C. Flores, W. Engels, Microsatellite instability in Drosophilaspellchecker 1 (MutS homolog) mutants, Proc. Natl. Acad. Sci.U.S.A. 96 (1999) 2964–2969.

[52] J. Yasuda, J.M. Navarro, S. Malkhosyan, A. Velazquez, R. Ar-ribas, T. Sekiya, M. Perucho, Chromosomal assignment of hu-man DNA fingerprint sequences by simultaneous hybridiza-tion to arbitrarily primed PCR products from human/rodentmonochromosome cell hybrids, Genomics 15 (1996) 1–8.

[53] S. Malkhosyan, J. Yasuda, J.L. Soto, T. Sekiya, J. Yokota, M.Perucho, Molecular karyotype (amplotype) of metastatic col-orectal cancer by unbiased arbitrarily primed PCR DNA finger-printing, Proc. Natl. Acad. Sci. U.S.A. 18 (1998) 10170–10175.

[54] T. Kohno, K. Morishita, H. Takano, D.N. Shapiro, J. Yokota,Homozygous deletion at chromosome 2q33 in human small-celllung carcinoma identified by arbitrarily primed PCR genomicfingerprinting, Oncogene 9 (1994) 103–108.

[55] J.N. Navarro, J.L. Jorcano, The use of arbitrarily primed poly-merase chain reaction in cancer research, Electrophoresis 20(1999) 283–290.

[56] B.J. Merrill, C. Holm, The RAD52 recombinational repair path-way is essential in pol30 (PCNA) mutants that accumulate smallsingle-stranded DNA fragments during DNA synthesis, Genet-ics 148 (1998) 611–624.

[57] M. Basik, D.L. Stoler, K.C. Kontzoglou, M.A. Rodrıguez-Bigas, N.J. Petrelli, G.R. Anderson, Genomic instability in spo-radic colorectal cancer quantitated by inter-simpled sequencerepeat PCR analysis, Genes, Chromosomes Cancer 18 (1997)19–29.

[58] D.X. Tishkoff, N. Filosi, G.M. Gaida, R.D. Kolodner, A novelmutation avoidance mechanism dependent on S. cervisiaeRAD27 is distinct from DNA mismatch repair, Cell 88 (1997)253–263.

[59] P.J. Lau, R.D. Kolodner, Transfer of the MSH2.MSH6 complexfrom proliferating cell nuclear antigen to mispaired bases inDNA, J. Biol. Chem. 278 (2003) 14–17.

[60] X. Yao, A.B. Buermeyer, L. Narayanan, D. Tran, S.M.Baker, T.A. Prolla, P.M. Glazer, R.M. Liskay, N. Arn-heim, Different mutator phenotypes in Mlh1-versus Pms2-deficient mice, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 6850–6855.

[61] P. Peltomaki, Deficient DNA mismatch repair: a common eti-ologic factor for colon cancer, Hum. Mol. Genet. 7 (2001)735–740.

[62] M.D. Schug, T.F.C. Mackay, C.F. Aquadro, Low mutation ratesof microsatellite loci in Drosophila melanogaster, Nat. Genet.15 (1997) 99–102.

[63] C. Scholetterer, R. Ritter, B. Harr, G. Brem, High mutationrates of a long microsatellite allele in Drosophila melanogasterprovides evidence for allele-specific mutation rates, Mol. Biol.Evol. 15 (1998) 1269–1274.

Germline genomic instability in PCNA mutants of Drosophila: DNA fingerprinting and microsatellite...

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