RESEARCH ARTICLES

Unscheduled DNA Replication Origin Activation atInserted HPV 18 Sequences in a HPV-18/MYC Amplicon

Chiara Conti,{ John Herrick, and Aaron Bensimon*

GenomicVision, 27 rue du Faubourg Saint Jacques,75014 Paris,France

Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood.

Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin

usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first

study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this

amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers.

The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the

same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher me-

dian frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent

increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replica-

tion origin activation at inserted HPV-18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subse-

quent overexpression of the MYC oncogene. This article contains Supplementary Material available at http://www.interscience.

wiley.com/jpages/1045-2257/suppmat. VVC 2007 Wiley-Liss, Inc.

INTRODUCTION

During S-phase, genome stability depends on

the faithful and complete duplication and trans-

mission of genetic information to the daughter

cells. Errors occurring during DNA replication are

known to be involved in chromosomal and gene

rearrangements. Amplification and deregulation of

cell cycle regulating genes, such as cyclins and tu-

mor suppressor genes, can lead to the develop-

ment of cancer and/or drug resistance. Despite the

importance of elucidating the sequence of events

leading to oncogene amplification, the mecha-

nisms generating such instabilities are still poorly

understood (Schar, 2001; Shiloh and Lehmann,

2004). Additional studies are necessary to improve

our understanding of DNA replication and the

anomalies that lead to amplifications in human

cancers.

In metazoa, DNA replication starts at multiple

origins of replication (Ori), from which two bidirec-

tional forks proceed and synthesize nascent DNA

strands (McWhinney and Leffak, 1990; Vassilev

and Johnson, 1990; Landry and Zannis-Hadjopou-

los, 1991). Origin positioning, spacing and timing,

together with fork progression, are the principle

parameters determining the kinetics of DNA repli-

cation (Fangman and Brewer, 1992; Sidorova and

Breeden, 2003). In normal S-phase, the DNA dam-

age ATM/ATR checkpoint is activated by feed-

back from active replicons to control origin firing

by down-regulating CDK2 and CDC7 kinases

(Miao et al., 2003; Marheineke and Hyrien, 2004;

Shechter et al., 2004), thus highlighting the impor-

tance of origin selection and activity for genome

integrity. In S. cerevisiae, initiation from fewer ori-

gins upon deletion of SIC1, a regulator of the G1/S

transition, is associated with gross chromosomal

rearrangements (GCR) (Lengronne and Schwob,

2002). Excessive origin activity also has severe con-

sequences for genome stability. Overexpression of

CDT1, a key initiation protein, as well as down-

regulation of the CDT1-inhibitor geminin, are

associated with rereplication, which activates the

ATM/ATR checkpoint response (Machida et al.,

2005; Saxena and Dutta, 2005). Therefore, the

number of origins itself appears to be crucial for

*Correspondence to: Aaron Bensimon, Genomic Vision, 27 rue duFaubourg Saint Jacques, 75014 Paris, France.E-mail: a.bensimon@ genomicvision.com

{Present address: Laboratory of Molecular Pharmacology, NCI,NIH, 37 Convent drive, Bldg. 37, Room 5066, Bethesda, MD, USA.

Supported by: Fondation Louis D, Institut de France.

Received 6 August 2006; Accepted 1 March 2007

DOI 10.1002/gcc.20448

Published online 19 April 2007 inWiley InterScience (www.interscience.wiley.com).

VVC 2007 Wiley-Liss, Inc.

GENES, CHROMOSOMES & CANCER 46:724–734 (2007)

the stability of the genome. On the other hand,

when replication forks experience a transient or

permanent replication arrest, they are converted

into substrates that may undergo recombinogenic

events, another potential source of genomic insta-

bility (Bosco and Haber, 1998; Hyrien, 2000;

McGlynn and Lloyd, 2002; Takeuchi et al., 2003;

Michel et al., 2004;). In budding yeast, in the ab-

sence of a specific and polar replication fork barrier,

the collision of the replication fork with the tran-

scription machinery was shown to generate tandem

direct amplification of rDNA and tRNA genes

(Deshpande and Newlon, 1996; Takeuchi et al.,

2003). In human cells, the rDNA locus was recently

shown to consist of repeated canonical and nonca-

nonical rearranged units (Lebofsky and Bensimon,

2005). Replication analysis within this region

revealed that fork progression is perturbed prefer-

entially at the noncanonical units, thus suggesting

that fork arrest is responsible for the rearrange-

ments. Since replication and transcription are spa-

tio-temporally separated, the fork barrier has been

suggested to be a safety mechanism to prevent po-

lymerase collision when segregation of transcrip-

tion and replication domains falters.

Despite the putatively known association

between incorrect DNA replication and genomic

instability (Schar, 2001), little has been done to

characterize precisely the altered regulation of ori-

gin firing and fork progression in amplified regions

in human cancers. However, elucidating these

aspects of DNA replication is important to fully

understand their roles in gene amplification.

In the present study we examine a spontaneous

amplicon, which we previously characterized in the

human cervical cancer IC1 (Herrick et al., 2005).

IC1 is an adenocarcinoma derived from glandular

cells where human papillomavirus 18 (HPV18)sequences are integrated nearby and coamplified

withMYC.HPV integration in the host genome is generally

accompanied by the disruption of the E2 protein

(Thierry et al., 1987), which represses transcrip-

tion from the E6 and E7 genes by binding to their

promoter. After integration, the disruption of E2

results in the expression of the oncoproteins E6and E7 (Farthing and Vousden, 1994; Scheffner et

al., 1994; Boyer, 1996; Mantovani and Banks,

2001; Munger et al., 2001). E6 promotes the deg-

radation of TP53, thus interfering with the G1/S

and G2/M checkpoints and reducing apoptosis

(Mantovani and Banks, 2001). E7 interferes with

the RRB pathway and induces unscheduled entry

into mitosis. Therefore, E6 and E7 are considered

to have a major role in the genomic instability that

is associated with the HPV18 infection. Since the

promoter overlaps with the viral origin of replica-

tion, the current understanding is that transcription

is repressed when DNA replication is active. E2

and the helicase E1, together with proteins of the

cellular replication machinery, are necessary for the

duplication of the virus. E1 binds to DNA with

very low specificity. However, the specificity of

binding to the origin of replication increases signif-

icantly when it interacts with E2, which binds to

three specific sites within the origin.

In IC1, sequences from the HPV18 and the

oncogene MYC constitute a cassette of *36 kb sta-

bly amplified at least 10-fold, which indicates that

the virus integrated into the cellular genome near

MYC prior to the amplification process (Fig. 1A)

(Herrick et al., 2005). As shown in Figure 1, HPV18sequences display a varying size that can be due to

rearrangements of the viral genome, as shown by

Peter et al. (2006). Quantitative PCR showed that

MYC was already amplified in the primary tumor

from which IC1 was derived (Peter et al., 2006).

PCR analysis showed that the viral genome is in-

terrupted in L1 and E1/E2 ORFs, whereas E6 and

E7 are intact (Herrick et al., 2005; Peter et al.,

2006), thus abrogating TP53 and RB1. The MYConcogene is overexpressed by about 10-fold, as

shown by RT-PCR (Peter et al., 2006). We used

this amplicon as a model to study origin usage and

fork progression in a repeated region. In IC1, the

organization of the region in tandem direct repeats

suggests a recombinogenic mechanism of amplifi-

cation based on rereplication or abnormal fork pro-

gression.

Origin activity and fork movement were moni-

tored and analyzed on individual DNA fibers

extracted from IC1 and stretched by molecular

combing (Bensimon et al., 1994; Lebofsky and

Bensimon, 2005). Since the original structure of a

region is maintained during the combing proce-

dure, the detection of replication signals com-

bined with the hybridization of fluorescent probes

can reveal the kinetics of DNA duplication in a

specific region. The replication kinetics of the

MYC/HPV amplicon compared with the reference

kinetics established at the whole genome scale

revealed anomalous origin activity and numerous

stalled forks. Because the region is highly tran-

scribed, we suggest that a collision between the

replication and the transcription polymerases

could induce fork arrest and recombination thus

resulting in the amplification of the MYC/HPV18cassette.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

725DNA REPLICATION KINETICS IN A HPV18/MYC AMPLICON

MATERIAL ANDMETHODS

Cell Culture and DNA Labeling

IC1 cells are derived from cervical cancer and

are naturally infected by HPV18 (Peter et al.,

2006). They were grown in RPMI media 1640

(Life Technologies, Gaithersburg, MD), supple-

mented with 5% FCS and a mixture of penicillin,

streptomycin and L-Glutamine (Life Technologies,

Gaithersburg, MD) to a final 13 concentration.

An asynchronous subconfluent population of

cells was pulse-labeled with 100 lM of Iodo-deox-

yuridine (IdU) (Sigma, St. Louis, MO) and 100 lMof Chloro-deoxyuridine (CldU) (Sigma, St. Louis,

MO) for 20 min each. Media containing the ana-

logues were preheated at 378C. The culture was

washed with PBS 13, preheated at 378C, betweenthe two analogues. At the end of the second pulse,

the cells were harvested, centrifuged and resus-

pended in PBS 13 at a final concentration of 5 3105/100 ll. An equal volume of 2% low melting

point liquid agarose was added to the suspension

of cells and the mix was aliquoted in PFEG molds

(170–3622, Bio-Rad, Herucules, CA). Once solid,

the plugs were washed over night with 1 mg/ml

proteinase K and 1% N-Lauryl Sarcosyl in 0.5 M

EDTA at 508C. Complete removal of digested pro-

teins and other degradation products was performed

by several gentle washings in TE. Protein-free

DNA plugs were stored in 0.5 M EDTA at 48C or

immediately used for combing.

DNAMolecular Combing

One plug or [1/4] of a plug were used for molec-

ular combing depending on the application to

study a specific locus or for a whole genome analy-

sis, respectively. The plug was melted at 708C for

20 min with 100 mM MES (pH 6.5). The solution

was maintained at 428C for 15 min and treated

overnight with 2 ll b-agarase (New England Biol-

abs, Ipswich, MA). The solution was dropped into

a teflon reservoir and DNA was combed on silan-

ized cover slips as previously described (Conti

et al., 2000).

Double Detection of IdU and CldU for Whole

Genome Analysis, Image Scanning, and Signal

Measurement

Combed DNA was denatured in 0.5 M NaOH

for 10 min with gentle shaking, washed several

times in PBS 13 and incubated with the primary

antibodies. All antibodies were diluted in a 1%

blocking solution (Boheringer, Ingelheim, Ger-

many), prepared in PBS 13, incubated in a humid

chamber and washed 33 3 min with PBS 13. First

step: 2/5 mouse anti-BrdU-FITC (Becton Dickin-

son, San Jose, CA) þ 2/5 rat anti-CldU (SeraLab,

Bolney, United Kingdom), incubated 1 hr at RT.

Second step: 1/25 donkey anti-mouse FITC

(Jackson Laboratory, Bar Harbor, ME) þ 1/25 don-

key anti-rat-594 (Molecular Probes, Carlsbad, CA),

Figure 1. DNA replication labeling and detection of origins in theMYC/HPV18 amplicon. (A) Coamplification of MYC and HPV-18 sequen-ces in the IC1 cell line. Red: HPV-18. Green: MYC. The virus and theoncogene are amplified as a single unit that includes a gap of undetectedchromosomal sequences. Bar ¼ 10 kb. (B) Schematic representation ofsignals deriving from equal pulse labeling with IdU and CldU. Replicationforks progress bidirectionally at the same rate from the origin andincorporate the analogues forming a symmetrical replicon. Upon detec-tion of IdU and CldU, three types of signals may be obtained: (1) a greenand red signal with a gap between the green segments corresponding toinitiations that occurred before the beginning of the pulse; (2) a dual-color signal with a continuous blue segment corresponding to originsthat fired during the first pulse; (3) a red signal corresponding to originsthat fired during the second pulse. A continuous red signal flanked bytwo blue signals is derived from the merge of two forks from adjacentorigins. The distance between the middle-point of two neighboring rep-licons is the inter-origin distance. The fork velocity is calculated bydividing the length of each fluorescent signal by the time of the pulse(dashed black arrows). (C) DNA replication ongoing in the amplicon,which is detected by the presence of HPV-18 probes in green. In eachpanel, probes alone are visible at the top, replication signals (in blue andin red) in the middle, the merge is at the bottom. According to thescheme in B, firings during the first and second pulse are detectable.Superposition of HPV probes with the middle-point of the replicationsignals indicates initiations occurring at viral sequences (white arrows:molecule a, b, and signal in the middle of molecule c). Firings at genomicsequences are also observable in the gap between the FISH probes(molecule c, signals on the left and on the right, and molecule d).

Genes, Chromosomes & Cancer DOI 10.1002/gcc

726 CONTI ET AL.

20 min. Mounting in Vectashield (Jackson Labora-

tory, Bar Harbor, ME). To detect BrdU incorpo-

rated into fully labeled DNA, the previous protocol

was used with the following modifications: First

step: 2/5 mouse anti-BrdU (Becton Dickinson, San

Jose, CA); Second step: 1/10 goat anti mouse-594

(Molecular Probes, Carlsbad, CA).

Probe Labeling and Hybridization on

Combed Molecules

The same DNA preparation was used to prepare

several surfaces with combed DNA. A part of those

surfaces was used to study the DNA replication

kinetics at the whole genome level, using the dou-

ble detection protocol described earlier. The

others were used for the DNA replication analysis

within the amplicon, which combines fluorescence

in situ hybridization (FISH) with a specific probe

for HPV18 and the detection of the replication

signals.

The probe to detect HPV18 sequences, a plas-

mid containing the full-length inactivated virus

(kindly provided by Dr. Gerard Orth), was labeled

by Random Priming (Invitrogen Life Technolo-

gies, Carlsbad, CA) with biotin-14-dUTP. 0.5 lg of

the biotinilated probe were precipitated with 33of 1 mg/ml Human Cot-1 DNA, dried and resus-

pended in the hybridization buffer (50% deionized

formamide, 23 SSC, 0.5% sodium dodecyl sulfate,

0.5% sarcosyl, 10 mM NaCl, and 33.3% blocking

solution [Boehringer, Ingelheim, Germany, block-

ing reagent prepared in PBS 13 with 0.05% Tween

20]).

Coverslips with combed DNA (stored at �208C)were dried and denatured for 20 min at RT in

0.5 M NaOH. Probes were denatured at 1008C for

5 min, incubated on ice for 5 min and hybridized

to the combed DNA in a humid chamber overnight

at 428C.

Triple Detection for Replication Analysis

Within a Specific Locus

Different fluorescent colors were used for the

analysis at the whole genome level and within the

amplicon. When possible, the blue fluorescence

was not used because it is more difficult to see at

the microscope. Therefore, for whole genome

studies IdU and CldU were detected in green and

red, respectively. However, to study the amplicon,

the blue color was introduced: the HPV18 probe

was detected in green, IdU in blue and CldU in

red. The protocol for the triple detection is

described.

Slides were washed with: (1) 50% deionized

formamide and 23 SSC, 3 3 5 min, with shaking

at room temperature (RT); (2) 23 SSC, 3 3 3 min,

with shaking at RT. The antibodies used to detect

probes and analogues were: (1) streptavidin Alexa-

Fluor 488 (Molecular Probes, Carlsbad, CA) 1/50,

mouse anti-BrdU (Becton Dickinson, San Jose,

CA) 2/5, Rat anti-BrdU (Harlan SeraLab, Oxon,

United Kingdom) 2/50, 1 hr at RT; (2) biotinilated

anti-streptavidin (Jackson Immunochemicals, West

Grove, PA) 1/50, cross-absorbed goat anti-mouse

Alexa Fluor 350 (Molecular Probes, Carlsbad, CA)

1/10, donkey anti-rat Alexa Fluor 594 (Molecular

Probes, Carlsbad, CA) 1/25, 20 min at 378C; (3)streptavidin AlexaFluor 488 (Molecular Probes,

Carlsbad, CA) 1/50, donkey anti-goat Alexa Fluor

350 (Molecular Probes) 1/10, donkey anti-rat Alexa

Fluor 594 (Molecular Probes, Carlsbad, CA) 1/25.

Antibodies were prepared in PBS 13 with 1%

blocking reagent (Boehringer, Ingelheim, Ger-

many). Incubations were carried out in a humid

chamber. After each step, slides were washed with

PBS 13, 3 3 3 min. After the final wash, slides

were mounted in anti-fading Vectashield (Jackson

Laboratory, Bar Harbor, ME).

Scanning and Statistical Analysis

Multiple slides were scanned to accumulate a

statistically reliable number of signals. Screening

was done with an epifluorescence microscope (Axio-

Plan, Zeiss, Le Pecq, France), using 403 objective,

connected to a CCD camera. Images were acquired

with Smart Capture 2.0. Measurements were per-

formed using CartographiX, a software program

developed in our lab. Removal of the background

was performed to enhance the distinction of the flu-

orescent signal from the background.

Data were inserted into an Excel spread sheet

and analyzed by KyPlot and SigmaPlot. For the

calculation of the fork velocity, we did not use: (1)

the red segments from merged forks; (2) the green

segments from Type II signals (Figs. 1A and 3) the

red segments from Type III signals (Fig. 1A). The

values of fork velocity and inter-origin distance

were calculated for each single molecule and plot-

ted as a frequency distribution.

Right versus left fork rates, for forks moving

from an origin, were determined for each single

replicon and their value is represented by a dot in

the scatter diagrams. The same procedure was

used to compare the IdU versus CldU track

belonging to a single fork. The data set is smaller

for the amplified region, which is significantly

shorter than the whole genome. Envelopes were

Genes, Chromosomes & Cancer DOI 10.1002/gcc

727DNA REPLICATION KINETICS IN A HPV18/MYC AMPLICON

drawn to define a pair of correlated forks when the

deviation of their ratio from the theoretical value 1

is less than 33%. Such a preset threshold allows for

future comparative studies with different cell lines,

growth conditions and different loci.

RESULTS

Origin Firing Occurs at Both Genomic and

Viral Sequences

The MYC/HPV18 amplicon in the IC1 cancer

derived cell line is a spontaneous and stable ampli-

con of about 350 kb, as observed on individual

DNA fibers by molecular combing (Herrick et al.,

2005). The stable maintenance of such a long

sequence likely depends on the presence of active

origins of replication. To map active origins in the

amplicon and to study their usage, asynchronous

exponentially-growing IC1 cells were sequentially

pulse-labeled with IdU and CldU, two thymidine

analogues, for an equal time of 20 min each. Cells

were immediately harvested and embedded in aga-

rose plugs to prepare a protein-free solution of high

molecular weight genomic DNA. This DNA was

then stretched by molecular combing (Bensimon

et al., 1994) with a constant and sequence-independ-

ent stretching factor (Michalet et al., 1997). Combed

DNA was hybridized with a biotin-labeled probe

belonging to HPV sequences (Onclercq et al., 1989)

and tricolor fluorescent detection was performed

(Lebofsky and Bensimon, 2005). IdU and CldU

were detected by fluorescent antibodies in blue and

red, respectively (Figs. 1B and 1C), whereas the

hybridized probes were detected in green. 60 firing

events could be observed over the 73 amplicons ana-

lyzed, half of which displayed only passive replica-

tion with one fork coming from the flanking regions.

Interestingly, initiations were observed at both

genomic and viral sequences although with different

frequencies (41/60 and 19/60, respectively) (Fig.

1C). This observation demonstrates that integrated

and rearranged viral sequences can be fully recog-

nized and licensed by the cellular replication ma-

chinery in the absence of a functional E2 (Herrick

et al., 2005).

Replication firing was observed at both genomic

and viral sequences. The present finding of foreign

viral DNA licensed for initiation following its inte-

gration into the cellular chromosome supports the

hypothesis that a relaxed sequence, together with

trans-acting and epigenetic factors, specifies the or-

igin in mammalian cells (Mechali, 2001). Unfortu-

nately, the experimental approach used here does

not allow for further investigation into whether

firings occurred at the known viral origin or at an

ectopic site licensed after integration.

Origins Are Activated in Clusters with Nearly

Synchronous Firing

Initiations occurring within the same chromatin

environment are expected to be activated synchro-

nously in clusters of 4–5 origins. It is therefore of

interest to examine the synchrony of origin firing

in an amplified region of the genome to determine

if this feature of the regulation of origin activation

is conserved.

In IC1, approximately the same proportion of

origins was activated in synchrony with an immedi-

ate neighbor both in the genome and in the ampli-

con (55 and 42%, respectively). Depending on the

color pattern of the replication signals obtained by

this approach, the time of firing can be accurately

determined: either prior to the addition of the ana-

logues or during the IdU or the CldU pulse (Fig.

1B). Analysis of 21 pairs of origins activated in the

amplicon showed that multiple firings occurred

during the same pulse or within a time-window of

*40 min (Fig. 2A).

The Frequency of Origin Activation Is Increased in

the Amplified Region

In the absence of clear criteria for establishing

the position of the origins, changes in origin usage

can be investigated according to their spacings

throughout the genome. DNA replication analysis

on combed DNA fibers allows for a quantitative

assessment of the origin distance when multiple

firings are detectable. The inter-origin distance

was measured at the whole genome scale (Herrick

et al., 2000; Lengronne et al., 2001; Anglana et al.,

2003; Lemaitre et al., 2005) to establish a reference

for the analysis of origin spacing within the ampli-

con. Genome wide analysis provided a median

(Md) value of 110 kb (n ¼ 111), versus 85.1 kb

measured in the amplified region (n ¼ 21) (Fig.

2B). Therefore, the median inter-origin distance is

smaller in the amplicon, indicating an increase in

the number of active origins. However, no signifi-

cant difference was found when comparing the

mean and distribution of the two samples (P >0.05 by the Kolmogorov–Smirnov test). Despite

the difference between the amplicon and the

genome sample sizes, the number of measure-

ments provided a feasible and comparable analysis

of the origin spacing.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

728 CONTI ET AL.

The MYC/HPV18 Amplicon Is Associated with

Stalled Forks

In a normal S-phase, the origin spacing was

shown to be positively and linearly correlated with

the fork velocity in human primary keratynocytes

and in the cervical cancer IC1 at the whole genome

level (Conti et al., unpublished data). These obser-

vations suggest the presence of a mechanism that

compensates for origin silencing with faster forks or,

conversely, with additional firing of dormant origins

following fork arrest (Anglana et al., 2003). An excess

of origins is licensed during G1. During S, once an

origin fires, it inhibits the neighboring origins from

firing unless a fork arrest takes place (DePamphilis,

1999; Anglana et al., 2003; Lebofsky et al., 2006).

Therefore, according to this model, the excess of ori-

gins activated in the MYC/HPV18 amplicon could be

a consequence of frequent fork arrests.

Fork velocity, together with origin distribution,

is the other major parameter governing replication

kinetics. On combed DNA molecules extracted

from IC1, the replication fork velocity was there-

fore measured in the amplified region and in the

genome. Figure 3A shows histograms of fork

speeds. The median value is 0.8 kb/min in the

amplicon (n ¼ 136) versus 1.5 kb/min in the ge-

nome (n ¼ 550) (P < 0.001 by the kolmogorov–

Smirnov test), which unambiguously indicates a

slower fork rate in the rearranged region. Interest-

ingly, this correlates with the closer origin-to-origin

distances found in the amplicon as compared with

the genome. These results are in agreement with

previous data showing a strict correlation between

initiation frequency and replication fork rates in

other cancer derived cell lines (Conti et al., unpub-

lished data) and suggest that an unknown mecha-

nism coordinates replication fork velocity with ori-

gin spacing.

During DNA synthesis, the replisome, or the

complex of proteins at the fork, can encounter

some obstacles that may perturb or block fork pro-

gression, such as secondary structures or large pro-

tein complexes bound to the DNA in addition to

DNA lesions. When DNA synthesis is not per-

turbed, forks emanating from one origin (referred to

in the text as outgoing forks) will move bidirectionally

Figure 2. Analysis of the origin dis-tribution and usage. (A) partial syn-chrony of initiations: all origins firedbefore the labeling (molecule 1), duringthe IdU blue pulse (molecule 2), duringthe CldU red pulse (molecule 3), orasynchronously but still within the 40min time-window (molecule 4). (B) His-tograms of the inter-origin distancemeasured in the genome (top) and inthe amplicon (bottom). The distributionprofiles indicate a shorter inter-origindistance in the amplicon B.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

729DNA REPLICATION KINETICS IN A HPV18/MYC AMPLICON

at the same speed, thus forming a symmetric repli-

con (Fig. 1B). Consequently, this will yield a posi-

tive linear correlation (R) between the two fork

velocities with a theoretical maximum value of

one. Replication fork arrest, on the other hand, will

result in asymmetric fork progression with little or

no correlation between the two fork rates (Fig.

3B).

Earlier we suggested that the additional initia-

tion events observed in the amplified region could

be a consequence of stalled forks. The lower fork

rate measured in the amplicon could be due to

slower processivity of the forks or, instead, to an

increased frequency of fork arrests. We therefore

performed an analysis on individual replicons and

compared the velocity of the left and right forks

moving from one origin located in the MYC/HPV18region and in the genome to evaluate the percent-

age of blocked forks. The result is presented as a

dot-plot where, to assess the frequency of asym-

metric replicons, we defined a threshold to discrim-

inate between correlated and noncorrelated fork

pairs (Fig. 3C). The threshold is shown as an enve-

lope. Data points within the envelope correspond

to pairs of forks moving bidirectionally at nearly

the same rate; conversely, data points lying outside

the envelope represent replicons where one of the

forks experienced an arrest and/or underwent a

change in its velocity. Forks emanating from one

origin were found to be linearly correlated both in

the amplicon and in the genome. However, the

correlation factor (R) is much weaker in the ampli-

con (r ¼ 0.38; P < 0.05; n ¼ 31) than in the genome

(r ¼ 0.86; P < 0.001; n ¼ 248). Indeed, the percent-

age of data points lying outside the threshold is

higher in the amplicon (74.2%) than in the genome

(16.9%), suggesting a higher frequency of fork

arrests.

Fork pausing and slowdown result in a different

amount of DNA that is replicated during the IdU

and CldU pulses. Therefore, in addition to the

analysis of forks emanating from one origin, the

comparison of the IdU and CldU tracks belonging

to an individual fork would also provide an esti-

mate of the frequency of fork arrests (Lebofsky

and Bensimon, 2005). Fork velocities during the

IdU and CldU pulses were compared and plotted

against each other (Figs. 4A and 4B). As shown pre-

viously, a threshold was defined to calculate the

number of dots outside the envelope, which corre-

spond to forks that experienced an arrest. A signifi-

cant positive linear correlation was calculated

Figure 3. Replication fork progression analysis. (A) Histograms ofthe fork velocity in the genome (top) and in the amplicon (bottom),show that forks progress slower in the amplified region. (B) Schematicrepresentation of asymmetric replicons resulting either from blockedforks (Type I signal) or from changes in fork velocity (Type II and 3 sig-nals). The vertical black line indicates a permanent replication fork bar-rier. Horizontal black lines show the segments that were measured andcompared to examine fork speed correlations. (C) Fork speeds from

the left and right of each replicon (scheme at the top of the figure)were plotted against each other for the genome (left) and the amplicon(right). The solid lines represent the envelope, which defines a thresh-old at 33%. The dashed line represents equal velocities for the left andthe right fork. Red dots correspond to replicons where one of the forkswas completely absent (see text and Fig. 4C). Data points outside theenvelope correspond to replicons that experienced a fork arrest.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

730 CONTI ET AL.

between the two tracks based on a genome-wide

analysis (r ¼ 0.80; P < 0.001; n ¼ 178) and an anal-

ysis of the amplified region (r ¼ 0.54; P < 0.001;

n ¼ 47). However, the strength of the correlation

was, again, weaker in the amplicon and the number

of data points lying outside the threshold is 55.3%,

versus the 32.5% occurring in the genome. There-

fore, the analysis of individual forks confirms the

presence of a significant number of stalled or re-

tarded forks in the MYC/HPV18 amplified region.

Replication signals with IdU and CldU tracks on

both sides of the midpoint suggest that the origin

is bidirectional. Sometimes, however, one of the

tracks was completely absent and the resulting sig-

nal was called a \unidirectional fork" (Fig. 4C).

The presence of a fork from a neighboring origin

during the whole-genome analysis, or the visualiza-

tion of the probes when the amplicon was analyzed,

excluded the possibility that the undetectable track

was due to DNA breakage caused by the combing

procedure. It is not possible to determine whether

the forks began from unidirectional origins or from

bidirectional origins that experienced a block before

the beginning of the IdU pulse. One unidirectional

fork was detected in the genome out of 248 repli-

cons analyzed (0.4%), whereas 6 out of 31 were

observed in the amplified region (19.4%). This ob-

servation corroborates previous results, which indi-

cate problems either at the origin or during fork

progression.

The MYC/HPV18 Region Is Completely Replicated

Before Entry into Mitosis

An unreplicated region entering mitosis may cre-

ate a dicentric chromosome that, when pulled apart

by the two poles on the mitotic spindle, breaks and

may lead to genomic aberrations. For this reason,

the intra-S and G2/M checkpoints arrest the cell

cycle before the onset of mitosis until the complete

genome is fully duplicated.

The frequent fork arrest observed in the MYC/HPV18 amplicon could result in a partial duplica-

tion of the region. Since checkpoints have a thresh-

old of sensitivity, an unreplicated portion of the

amplicon may be insufficient to alert the surveil-

lance mechanisms. Additionally, in IC1, the normal

checkpoint activity is abrogated by the degradation

of TP53 and RB1 (Boyer et al., 1996; Mantovani

Figure 4. Replication fork arrest analysis. (A) Replication fork arrestin the MYC/HPV18 amplicon. HPV18 probes were detected in green,IdU in blue, and CldU in red. The absence of the red track, correspond-ing to CldU incorporation during the second pulse, suggest a perma-nent fork block, represented by the white vertical line in molecules 1and 2. In molecule 3, the different blue signal length on the left and onthe right may indicate a slowdown, whereas a block or a slowdown can-not be distinguished in molecules 4 and 5. (B) Fork speed from the first(IdU) and second (CldU) pulse were plotted against each other, for thegenome (top) and the amplicon (bottom). The solid lines represent the

envelope, which defines a threshold at 33%. The dashed line representsequal velocities for the left and the right fork. (C) Two examples of uni-directional forks, as a result of a permanent fork arrest. At the top,scheme of a bidirectional replicon flanked by a fork. HPV18 probeswere detected in green, IdU in blue, and CldU in red. Sometime, one ofthe forks was completely absent. In all cases, the presence of the probesignals and of an incoming fork from a neighboring origin guaranteed anintact DNA combed molecule. Therefore, the solitary fork could resultfrom either a unidirectional origin or from a bidirectional repliconwhere one fork experienced a permanent block before the labeling.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

731DNA REPLICATION KINETICS IN A HPV18/MYC AMPLICON

and Banks, 2001; Munger et al., 2001). To ascertain

whether the region was totally replicated, we did a

24 hr-pulse with bromo-deoxyuridine (BrdU) in an

asynchronous population of IC1 cells. Combed

DNA was hybridized with a probe belonging to

HPV18 and dual-color detection was performed

with fluorescent antibodies. The probes were

detected in green, whereas the BrdU was detected

in red. Images show that the MYC/HPV18 amplicon

is fully duplicated (Supplementary Figure; Supple-

mentary material for this article can be found at

http://ww.interscience.wiley.com/jpages/1045-2257/

suppmat). This suggests that the genomic rear-

rangements are unlikely to have occurred during

mitosis.

DISCUSSION

Gene rearrangements and amplifications are a

hallmark of cancer cells. Therefore, understanding

the mechanisms behind these instabilities would

help in studying the steps that lead to neoplastic

transformation. During S-phase, genome duplica-

tion is the major source of chromosomal instability,

but the changes in the DNA replication program

that drive gene amplification are still not fully

understood. Molecular details of the replication

kinetics in rearranged regions would help to pro-

vide clues about the role of DNA replication in the

amplification process. In this article, we provide

the first analysis of DNA replication kinetics

involving a spontaneously amplified MYC/HPV18region organized in tandem direct repeats in a

human cervical cancer. We have used the replica-

tion kinetics established at the whole genome level

as a reference for a comparative study to ascertain

whether the normal kinetics were maintained dur-

ing duplication of the amplified region and

whether any abnormalities may play a role in the

amplification process.

Since the number of origins is critical for

genome stability (Lengronne and Schwob, 2002;

Saxena and Dutta, 2005), we analyzed the origin

usage and distribution along the amplicon. We

found that multiple firings occur within *40 min,

in agreement with previous results obtained in the

human rDNA locus (Caburet et al., 2005; Lebofsky

and Bensimon, 2005). Therefore, our observations

show that the supplemental and unexpected genetic

material contained in the amplicon is duplicated

according to the same temporal initiation program

Figure 5. Schematic representation of the steps leading to the MYC/HPV18 amplification. 1st cycle: Upon viral integration near MYC (a), rep-lication can initiate from both genomic and viral sequences. In a TP53-depleted background, this change in the origin distribution may triggerrereplication (b and c) that generates the so-called onion skin structureand double strand breaks. The DNA damage might be repaired by non-homologous recombination (d), thus generating twoMYC/HPV18 copiesin tandem (e). 2nd cycle: Integration of HPV18 induces high transcrip-tion of MYC and viral genes. Because of the additional replication firingsfrom viral sequences (f), there are sufficient forks traveling in the region

to collide with the transcription polymerase. The resulting DNA nick(g) can be repaired by searching for the homologous sequence on thesister chromatid (h), which induces a further amplification of the MYC/HPV18 unit. (i) The cassette searches for its homologous sequenceamong the two copies on the other strand and forms a duplex with anupstream copy. The replication fork duplicates the leftward copyand the double-strand-break that is generated can be repaired byrecombination. As a consequence, one strand will gain one copy of thecassette.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

732 CONTI ET AL.

as was observed for a normal multicopy locus such

as the rDNA region.

A slight increase in the number of active origins

was observed in theMYC/HPV18 region. The intro-

duction of the additional and unscheduled firings

from viral sequences might have perturbed the

normal regulation of the MYC origin. A TP53-de-

pendent checkpoint was shown to prevent rereplica-

tion (Vaziri et al., 2003; Claycomb and Orr-Weaver,

2005), which is associated with extensive DNA dam-

age and double strand breaks (DSB), a favored sub-

strate for recombinogenic events (Green and Li,

2005). In IC1, FACS analysis did not show evidence

of rereplication at the genome level (data not

shown), but the approach is not sensitive enough to

detect infrequent events of refiring in a small portion

of the genome. Illegitimate recombination between

imperfectly homologous sequences is also controlled

in a TP53-dependent manner (Akyuz et al., 2002).

In IC1, E6, and E7 are actively transcribed (Herrick,

et al., 2005), with subsequent loss of TP53 activity.

Therefore, origins might undergo successive refir-

ings, as previously suggested for the polyomavirus

(Py) genomes and other viruses integrated into the

cellular chromosome and organized in a head-to-tail

ladder structure (Syu and Fluck, 1997).

Analysis of the fork progression revealed a high

percentage of fork arrests in the amplicon. Stalled

and arrested forks can be converted into a DSB,

which is an ideal substrate for recombination-

mediated repair. TP53 was shown to protect the

genome from replication-associated DSB and to

suppress homologous recombination (Kumari

et al., 2004). To explain the linear array of MYC/HPV18 cassettes, two events might occur after

HPV integration in the absence of TP53: (1) an

occasional refiring that creates an onion-skin struc-

ture, subsequently resolved by illegitimate recom-

bination, or (2) a collision of the replication and

transcription polymerases, thus inducing replica-

tion fork arrest and recombination (Fig. 5) (Take-

uchi et al., 2003). MYC expression is 10-fold higher

than in normal tissues (Peter et al., 2006), with an

estimated 10 MYC copies per cell (Herrick et al.,

2005). In addition, transcription of E6 and E7 viral

genes is usually 5- to 10-fold higher than other cel-

lular genes. Therefore, a transcription polymerase

every 100 kb can be expected. Considering the

inter-origin distance of 96 kb measured here and

an average distance of 36 kb between two MYCcopies, a sufficient number of replication forks and

transcription factories can travel along the MYC/HPV18 amplicon and might collide, as suggested

by the second hypothesis. This supports the mech-

anism based on an abnormal replication program

previously suggested by the authors (Herrick et al.,

2005). In addition to the \gain of copy" by illegiti-

mate recombination, we propose a mechanism

based on the search for homologous sequences.

Unreplicated DNA can break and generate tan-

dem inverted repeats by the break-fusion-bridge

(BFB) during mitosis (McClintock, 1951; Hellman

et al., 2002). It was also suggested that during the

first stages of infection a functional E2 protein may

be responsible for the amplification of the virus by

BFB (Van Tine et al., 2004). However, the BFB

mechanism is not compatible with a structure of

direct repeats as observed in IC1. Since the ampli-

con contains active origins and is fully duplicated,

it can therefore be stably propagated.

The periodic structure of the amplicon with sta-

ble MYC/HPV18 cassettes indicates that viral inte-

gration occurred before the amplification (Herrick

et al., 2005). Since there is no specific sequence tar-

geted by the virus, it is unlikely that viral integra-

tion took place at each additional MYC copy after

they were amplified. This further strengthens the

hypothesis that an aberrant DNA replication pro-

gram is the cause of the amplification after integra-

tion of the virus into the host genome. Additional

studies on more samples from cervical cancers will

be necessary to investigate whether the results

shown here are general to HPV18-infected cancers.

ACKNOWLEDGMENTS

The authors thank Gabriel Eichler for helpful

discussions and corrections to the English.

REFERENCES

Akyuz N, Boehden GS, Susse S, Rimek A, Preuss U, ScheidtmannKH, Wiesmuller L. 2002. DNA substrate dependence of p53-mediated regulation of double-strand break repair. Mol Cell Biol22:6306–6317.

Anglana M, Apiou F, Bensimon A, Debatisse M. 2003. Dynamics ofDNA replication in mammalian somatic cells: Nucleotide pool mod-ulates origin choice and interorigin spacing. Cell 114:385–394.

Bensimon A, Simon A, Chiffaudel A, Croquette V, Heslot F, Bensi-mon D. 1994. Alignment and sensitive detection of DNA by amoving interface. Science 265:2096–2098.

Bosco G, Haber JE. 1998. Chromosome break-induced DNA repli-cation leads to nonreciprocal translocations and telomere capture.Genetics 150:1037–1047.

Boyer SN, Wazer DE, Band V. 1996. E7 protein of human papillomavirus-16 induces degradation of retinoblastoma protein throughthe ubiquitin-proteasome pathway. Cancer Res 56:4620–4624.

Caburet S, Conti C, Schurra C, Lebofsky R, Edelstein SJ, BensimonA. 2005. Human ribosomal RNA gene arrays display a broad rangeof palindromic structures. Genome Res 15:1079–1085.

Claycomb JM, Orr-Weaver TL. 2005. Developmental gene amplifi-cation: Insights into DNA replication and gene expression.Trends Genet 21:149–162.

Conti C, Caburet S, Bensimon A. 2000. Molecular combing. In: Cur-rent Protocols in Cytometry, Vol. 1, unit 10, John Wiley and Sons,New York.

DePamphilis ML. 1999. Replication origins in metazoan chromo-somes: Fact or fiction? Bioessays 21:5–16.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

733DNA REPLICATION KINETICS IN A HPV18/MYC AMPLICON

Deshpande AM, Newlon CS. 1996. DNA replication fork pausesites dependent on transcription. Science 272:1030–1033.

Fangman WL, Brewer BJ. 1992. A question of time: Replication ori-gins of eukaryotic chromosomes. Cell 71:363–366.

Farthing AJ, Vousden KH. 1994. Functions of human papillomavirusE6 and E7 oncoproteins. Trends Microbiol 2:170–174.

Green BM, Li JJ. 2005. Loss of rereplication control in Saccharomycescerevisiae results in extensive DNA damage. Mol Biol Cell16:421–432.

Hellman A, Zlotorynski E, Scherer SW, Cheung J, Vincent JB,Smith DI, Trakhtenbrot L, Kerem B. 2002. A role for commonfragile site induction in amplification of human oncogenes.Cancer Cell 1:89–97.

Herrick J, Stanislawski P, Hyrien O, Bensimon A. 2000. Replicationfork density increases during DNA synthesis in Xenopus laevis eggextracts. J Mol Biol 300:1133–1142.

Herrick J, Conti C, Teissier S, Thierry F, Couturier J, Sastre-GarauX, Favre M, Orth G, Bensimon A. 2005. Genomic organization ofamplified MYC genes suggests distinct mechanisms of amplifica-tion in tumorigenesis. Cancer Res 65:1174–1179.

Hyrien O. 2000. Mechanisms and consequences of replication forkarrest. Biochimie 82:5–17.

Kumari A, Schultz N, Helleday T. 2004. p53 protects from replica-tion-associated DNA double-strand breaks in mammalian cells.Oncogene 23:2324–2329.

Landry S, Zannis-Hadjopoulos M. 1991. Classes of autonomouslyreplicating sequences are found among early-replicating monkeyDNA. Biochim Biophys Acta 1088:234–244.

Lebofsky R, Bensimon A. 2005. DNA replication origin plasticityand perturbed fork progression in human inverted repeats. MolCell Biol 25:6789–6797.

Lebofsky R, Heilig R, Sonnleitner M, Weissenbach J, Bensimon A.2006. DNA replication origin interference increases the spacingbetween initiation events in human cells. Mol Biol Cell 17:5337–5345.

Lemaitre JM, Danis E, Pasero P, Vassetzky Y, Mechali M. 2005.Mitotic remodeling of the replicon and chromosome structure.Cell 123:787–801.

Lengronne A, Pasero P, Bensimon A, Schwob E. 2001. Monitoring Sphase progression globally and locally using BrdU incorporationin TK(þ) yeast strains. Nucleic Acids Res 29:1433–1442.

Lengronne A, Schwob E. 2002. The yeast CDK inhibitor Sic1 pre-vents genomic instability by promoting replication origin licens-ing in late G(1). Mol Cell 9:1067–1078.

Machida YJ, Hamlin JL, Dutta A. 2005. Right place, right time, andonly once: Replication initiation in metazoans. Cell 123:13–24.

Mantovani F, Banks L. 2001. The human papillomavirus E6 proteinand its contribution to malignant progression. Oncogene 20:7874–7887.

Marheineke K, Hyrien O. 2004. Control of replication origin densityand firing time in Xenopus egg extracts: Role of a caffeine-sensi-tive, ATR-dependent checkpoint. J Biol Chem 279:28071–28081.

McClintock B. 1951. Chromosome organisation and genetic expres-sion. Cold Spring Harbor Symp Quant Biol 16:13–47.

McGlynn P, Lloyd RG. 2002. Recombinational repair and restart ofdamaged replication forks. Nat Rev Mol Cell Biol 3:859–870.

McWhinney C, Leffak M. 1990. Autonomous replication of a DNAfragment containing the chromosomal replication origin of thehuman MYC gene. Nucleic Acids Res 18:1233–1242.

Mechali M. 2001. DNA replication origins: From sequence specific-ity to epigenetics. Nat Rev Genet 2:640–645.

Miao H, Seiler JA, Burhans WC. 2003. Regulation of cellular andSV40 virus origins of replication by Chk1-dependent intrinsic andUVC radiation-induced checkpoints. J Biol Chem 278:4295–4304.

Michalet X, Ekong R, Fougerousse F, Rousseaux S, Schurra C,Hornigold N, van Slegtenhorst M, Wolfe J, Povey S, BeckmannJS, Bensimon A. 1997. Dynamic molecular combing: Stretchingthe whole human genome for high-resolution studies. Science277:1518–1523.

Michel B, Grompone G, Flores MJ, Bidnenko V. 2004. Multiplepathways process stalled replication forks. Proc Natl Acad SciUSA 101:12783–12788.

Munger K, Basile JR, Duensing S, Eichten A, Gonzalez SL, GraceM, Zacny VL. 2001. Biological activities and molecular targets ofthe human papillomavirus E7 oncoprotein. Oncogene 20:7888–7898.

Onclercq R, Lavenu A, Cremici C. 1989. Pleiotropic derepression ofdevelopmentally regulated cellular and viral genes by MYC proto-oncogene products in undifferentiated embryonal carcinoma cells.Nucleic Acids Res 17:735–753.

Peter M, Rosty C, Couturier J, Radvanyi F, Teshima H, Sastre-Garau X. 2006. MYC activation associated with the integration ofHPV DNA at the MYC locus in genital tumors. Oncogene25:5985–5993.

Saxena S, Dutta A. 2005. Geminin-Cdt1 balance is critical forgenetic stability. Mutat Res 569:111–121.

Schar P. 2001. Spontaneous DNA damage, genome instability, andcancer—When DNA replication escapes control. Cell 104:329–332.

Scheffner M, Romanczuk H, Munger K, Huibregtse JM, Mietz JA,Howley PM. 1994. Functions of human papillomavirus proteins.Curr Top Microbiol Immunol 186:83–99.

Shechter D, Costanzo V, Gautier J. 2004. ATR and ATM regulatethe timing of DNA replication origin firing. Nat Cell Biol 6:648–655.

Shiloh Y, Lehmann AR. 2004. Maintaining integrity. Nat Cell Biol6:923–928.

Sidorova JM, Breeden LL. 2003. Precocious G1/S transitions andgenomic instability: The origin connection. Mutat Res 532:5–19.

Syu LJ, Fluck MM. 1997. Site-specific in situ amplification of theintegrated polyomavirus genome: A case for a context-specificover-replication model of gene amplification. J Mol Biol 271:76–99.

Takeuchi Y, Horiuchi T, Kobayashi T. 2003. Transcription-depend-ent recombination and the role of fork collision in yeast rDNA.Genes Dev 17:1497–1506.

Thierry F, Carranca AG, Yaniv M. 1987. Elements that control thetranscription of genital human Papillomavirus HPV18. CancerCells 5:23–32.

Van Tine BA, Dao LD, Wu SY, Sonbuchner TM, Lin BY, Zou N,Chiang CM, Broker TR, Chow LT. 2004. Human papillomavirus(HPV) origin-binding protein associates with mitotic spindles toenable viral DNA partitioning. Proc Natl Acad Sci USA 101:4030–4035.

Vassilev L, Johnson EM. 1990. An initiation zone of chromosomalDNA replication located upstream of the MYC gene in proliferat-ing HeLa cells. Mol Cell Biol 10:4899–4904.

Vaziri C, Saxena S, Jeon Y, Lee C, Murata K, Machida Y, Wagle N,Hwang DS, Dutta A. 2003. A p53-dependent checkpoint pathwayprevents rereplication. Mol Cell 11:997–1008.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

734 CONTI ET AL.