Research Communication

Specific Replication Factors are Targeted by Different GenotoxicAgents to Inhibit Replication

Aparna Sharma,* Ananya Kar,* Manpreet Kaur,* Sourabh M. Ranade, Aparna Sankaran,

Shashank Misra, Kanchan Rawat and Sandeep Saxena*National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India

Summary

When mammalian cells experience DNA damaging stress,they block DNA replication to avoid erroneous replication ofthe damaged template. The cells that are unable to respond toDNA damage continue faulty DNA replication that results inincorporation of genomic lesions. To understand the regulationof replication machinery during stress, systemic studies havebeen carried out but they have been restricted to the evaluationof the mRNA levels and therefore have not been able to identifypost-transcriptional changes, vital for immediate blocking of theprogressing DNA replication. We have recently discovered thatan essential replication factor is downregulated by radiationstress. In this study, we have carried out a systematic evaluationof protein levels of entire replication apparatus after differenttypes of DNA damage. We report that, independent of the sta-tus of p53 and retinoblastoma protein, mammalian cells choosetargets that are essential for prereplication, preinitiation, andelongation phases of replication. We imposed different kinds ofstress to discern whether similar or unique responses areinvoked, and we propose a model for inhibition of replicationmachinery in which mammalian cells target specific essentialreplication factors based on the experienced stress. � 2010

IUBMB

IUBMB Life, 62(10): 764–775, 2010

Keywords DNA replication; genomic stability; stress response; DNA

damage; replication proteins.

INTRODUCTION

DNA replication initiates at specific regions on the chromosome

called origins of replication (1). Origin recognition complex, which

consists of six subunits (ORC1-6), binds to the origins of replica-

tion, which is followed by the recruitment of replication initiators,

Cdc6 and Cdt1 (2, 3). The putative helicase, MCM 2-7 complex,

follows next and once it gets bound along with ORC, Cdc6, and

Cdt1, it is termed as prereplication complex (Fig. 1) (1, 4). There is

a burst of kinase activity (cyclin E/cdk2 and DBF4-Cdc7) at the G1/

S transition with the simultaneous recruitment of essential replica-

tion protein, Mcm10 (5, 6). In the recent years, our understanding

of the replication initiation process has advanced significantly, and it

is demonstrated that a complex of proteins, GINS, is recruited to

the origins of replication at this stage (7, 8). The binding of GINS

to the chromosomes is interdependent on the loading of TopBP1,

Cdc45-Sld3, and DNA polymerase epsilon-Sld2 complexes (9, 10).

The successful recruitment of these factors leads to the recruitment

of the single-stranded DNA binding protein, RPA, to stabilize the

unwound DNA (11). DNA primase then synthesizes RNA primers,

which are elongated by DNA polymerase alpha and subsequently

removed by RNase H (12). Clamp loader, replication factor C, then

mediates the loading of the sliding clamp, PCNA, which is followed

by binding of polymerase epsilon and delta to the primer-template

junction for carrying out DNA synthesis (13, 14).

When mammalian cells encounter DNA damage, various pro-

tective mechanisms are invoked, termed as checkpoint pathways,

to halt the DNA replication and repair the damage (15, 16).

Broadly, there are two major molecular cascades that get activated

depending on the kind of DNA damage. Ionizing radiation, which

causes double strand breaks, activate the Ataxia telangiectasia

mutated (ATM) kinase whereas UV radiation and DNA modifying

chemicals, which lead to replication blocks, primarily activate

Ataxia telangiectasia and Rad3 related (ATR) kinase. Oxidative

stress leads to base modifications and strand breaks activating the

various arms of DNA damage checkpoint pathways (17). ATM

phosphorylates several key proteins including p53, CHK2,

Additional Supporting Information may be found in the online ver-

sion of this article.

*These authors contributed equally to this work.

Received 2 July 2010; accepted 18 August 2010

Address correspondence to: Sandeep Saxena, National Institute of

Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India. Tel: 91-

11-26703726. Fax: 91-11-26742125. E-mail: sandeep@nii.res.in

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.380

IUBMB Life, 62(10): 764–775, October 2010

BRCA1, and H2AX leading to cell cycle arrest and apoptosis (18,

19). However, ATR along with its partner protein ATRIP, phos-

phorylates Chk1, leading to a cell cycle arrest (20, 21). Chk1

phosphorylates Cdc25c phosphatase, which leads to its sequestra-

tion in cytoplasm, inhibiting Cdk1 and consequently causing a

cell-cycle block (22, 23). The requirement of Chk1, ATM, and

ATR checkpoint kinases have been demonstrated for the degrada-

tion of Cdc25a protein phosphatase, which leads to the activation

of the intra S phase checkpoint (24). Apart from the regulation of

phosphorylation and protein turnover of Cdc25a by Chk1, the inhi-

bition of its transcription is mediated by p53. The p53 plays a

major role in maintaining the integrity of the genome following

various forms of insults and its mutation or deletion is observed in

almost half of human cancers (25). In response to DNA damage

transduction signal from ATM/ATR kinases, p53 mediates either a

G1 arrest or apoptosis (26). Therefore, based on the nature of dam-

age and phase of cell cycle, the cell utilizes a maze of protective

checkpoint proteins to accomplish a cell cycle block till the dam-

aged DNA is repaired.

Cells that lack checkpoint functions are unable to respond to

DNA damage and continue faulty replication resulting in

genomic instability. Therefore it is essential to understand the

regulation of replication machinery during stress (27). Different

studies have established that on perceiving stress, a dose de-

pendent regulation of transcription occurs and many replication

genes are repressed within 12–24 h to stall replication (28–30).

Although the transcriptional block would eventually inhibit the

downregulated proteins, it is unlikely to lead to an immediate

cessation of DNA replication. In this study, we aimed to iden-

tify the proteins that are downregulated immediately following

DNA damage. We have systematically analyzed the stability of

23 replication factors ranging from the replication initiator to

processive DNA polymerase epsilon after exposure to five dif-

ferent forms of DNA damage. We also investigated whether dif-

ferent forms of stress share the same replication target or inhibit

unique targets for arresting replication. In summation, we pro-

pose a model for inhibition of replication machinery in which

mammalian cells target specific essential replication factors

based on the stress experienced.

MATERIALS AND METHODS

Cell Culture, Antibodies, and Exposure to Stress

Dulbecco’s modified Eagle’s medium (DMEM) medium

supplemented with 10% (v/v) heat-inactivated fetal calf serum

(Biological Industries, Kibbutz Beit-Haemek, Israel), 100 units/

mL penicillin and 100 lg/mL streptomycin (Invitrogen, Carls-

bad, CA) was used to maintain HeLa and U2OS cell lines (31–

Figure 1. A simplified model for replication initiation in eukaryotes. The replication process initiates with the recruitment of the

six-subunit origin recognition complex (ORC) to the replication origins during the M phase. Replication initiators, Cdc6 and Cdt1,

and MCM helicase are next recruited to form the prereplicative complex (pre-RC) in M to G1 phase of the cell cycle. At the G1/S

transition there is an increase in activity of cyclin dependent kinase (CDK) and Dbf4-dependent kinase (DDK) and recruitment of

Mcm10 to the origins of replication. This leads to the simultaneous association of Cdc45 and GINS complex, forming the preinitia-

tion complex (pre-IC) and unwinding of the replication origins. Single-stranded DNA binding protein, RPA, stabilizes the unwound

strands of DNA and DNA polymerase alpha-primase (POL a) is recruited to synthesize the RNA primers. Subsequently RFC medi-

ates the loading of the sliding clamp, PCNA which is followed by binding of polymerase epsilon (POL e) and delta (POL d) to the

primer-template junction for carrying out DNA synthesis.

765STRESS INDUCED PROTEOLYSIS OF REPLICATION FACTORS

33). The antibodies used for immunoblotting (IB) and indirect

immunofluorescence (IF) studies are described in the supple-

mental information. U2OS and HeLa cells were exposed to 100

and 200 J/m2 of 254 nm UV-radiation respectively and 50 Gy

gamma-radiation and harvested after 1 h and 4 h for immuno-

blotting and immunofluorescence. After incubation with a com-

bination of genotoxic agents including bleomycin (25 mU/ml),

cisplatin (10 lM), doxorubicin (10 lM) and cyclophosphamide

(100 lM), HeLa and U2OS cells were harvested after 1 h and 4

h. Bleomycin causes strand breaks, cyclophosphamide and cis-

platin, cause DNA cross-linking and doxorubicin intercalates in

the DNA double helix causing DNA strand breaks (34–37). To

block the cell cycle at S-phase and induce the checkpoint path-

way, HeLa and U2OS cells were harvested 1, 2, and 8 h post

incubation in media supplemented with 1.3 mM hydroxyurea.

For inducing oxidative damage, HeLa and U2OS cells were

incubated in the presence of 2.5 mM hydrogen peroxide for 1

h, released in drug-free medium and harvested 3 h post release

(38, 39).

Immunoblotting and Indirect Immunofluorescence

Following the treatment with genotoxic agents, the cells

were harvested in SDS sample buffer and equal amount of pro-

tein was resolved on SDS-polyacrylamide gel followed by trans-

fer on nitrocellulose membrane for immunoblotting (40, 41).

The membrane was blocked with 5% non-fat milk powder in

Tris-buffered saline and after incubation with the specific pri-

mary antibody (1:1000 or 1:2000 dilution) and secondary anti-

body (1:2000 dilution), the results were assayed using the

Enhanced Chemiluminescence method (42). The level of repli-

cation proteins after exposure to different forms of stress were

quantitated using the Quantity One Software (version 4.6.3;

Bio-Rad). We have calculated the mean and standard deviation

of two independent experiments and reported the same in the

results. For indirect immunofluorescence studies, HeLa and

U2OS cells were grown on glass coverslips in DMEM supple-

mented with 10% fetal bovine serum (FBS) (43). The cells on

the coverslips were then exposed to different stresses followed

by fixing with 4% formaldehyde in PBS for 10 min and then

permeabilization with 0.2% Triton X-100 in PBS for 5 min.

Later, the cells were blocked with 10% FBS in PBS with 0.1%

Tween-20. The cells were then stained with primary antibody

(1:100 or 1:500 dilution) for an hour followed by either fluores-

cein isothiocyanate-conjugated anti-rabbit or anti-mouse anti-

body or Alexa-594 conjugated anti-goat antibody (1:500 dilu-

tion) at room temperature. The coverslips were then mounted

with vectashield mounting reagent with DAPI and viewed under

the Nikon TE2000-S inverted fluorescence microscope. Images

were captured on Evolution VF (Media Cybernetics) 12-bit

color digital camera using the ‘Q capture Pro’ software and

contrast enhancements were identically done for all the images

of a particular antibody/ protein in an experiment. Images

obtained with different antibodies were pasted in Adobe Photo-

shop after reduction to approximately 40% of the original size.

Some images were captured using the Zeiss LSM 510 confocal

microscope and pasted in Adobe Photoshop after reduction to

approximately 75% of the original size. Finally, all images were

assembled and proportionally reduced to 20% to fit in a

230 mm height X 178 mm width page.

RNA Interference (RNAi) Silencing

HeLa cells were seeded in 6-cm cell culture dishes and

transfected with specific 40-80 nM small inhibitory RNA

(siRNA) using Lipofectamine 2000 reagent (Invitrogen) for 2–3

consecutive days (44, 45). Twenty-four hours after the last

transfection, the cells were harvested and protein levels were

checked by immunoblotting. For immunofluorescence analysis,

cells were grown on coverslips for 12–24 h after the last trans-

fection and processed for immunofluorescence according to the

procedure described above.

RESULTS

Regulation of Replication Apparatus on Exposure to UV,Gamma Radiation, and Genotoxic Chemicals

Our objective was to discern the effects of genotoxic agents

on proteins, which function at different steps in the replication

process. We used HeLa and U2OS cells, which have been exten-

sively used for similar studies (46–48). HeLa cells are infected

with human papillomavirus whose products E7 and E6, target the

retinoblastoma (Rb) and p53 protein, respectively and therefore

these cells are deficient in activities of these key checkpoint

proteins, whereas U2OS cells express wild type p53 and Rb

(31–33). Following DNA damage, p53 activates many down-

stream proteins to activate the cell-cycle checkpoints, DNA

repair, senescence, apoptosis, and the surveillance of genomic in-

tegrity (49–51). Functional interactions between p53 and canoni-

cal checkpoint proteins such as ATM, ATR, H2AX, and NBS1

are essential for checkpoint response to DNA damage (26, 52). It

has been observed that deficient activity of p53 leads to enhanced

sensitivity to DNA damage manifesting in many forms of human

tumors (25, 53). Therefore, we choose these two cell lines, which

have inherent differences in responding to stress.

In this study, we have utilized a diverse range of genotoxic

agents such as ionizing gamma radiation, which causes double

strand breaks, UV radiation, which leads to formation of pyrim-

idine dimers causing a replication block, and a combination of

DNA-damaging chemicals which cause different forms of dam-

age to induce stress in asynchronously growing HeLa and

U2OS cells. We used high doses of radiation (100–200 J/m2

UV and 50 Gy gamma radiation) to detect even minor changes

in replication apparatus as lower doses may not reveal some of

the stress-induced responses. Further, we tested the stability of

downregulated replication proteins at different doses of geno-

toxic agents (Fig. 4A). We have examined the changes in the

stability of downregulated replication proteins at 1 h and 4 h

766 SHARMA ET AL.

post-stress. Replication factor Cdt1 is degraded in few minutes

after irradiation and therefore we tested the stability of replica-

tion factor at 1 h time point to identify immediate response to

damage (46). Degradation of replication factor Mcm10 was

observed to be temporally separated from instantaneous Cdt1

downregulation and therefore we tested stability at 4 h time-

point to identify changes in replication apparatus that are slower

(54). The canonical checkpoint kinase, Chk1, was phosphoryl-

ated at Serine 345 after exposure to all the genotoxic agents,

confirming DNA damage (Figs. 2A and 2B) (55). We observed

Figure 2. Regulation of replication proteins in mammalian cells following different forms of DNA damage. HeLa (A) and U2OS

(B) cells were exposed to 100 or 200 J/m2 UV (UV-rad), 50 Gy gamma radiation (c-rad) and DNA damaging chemicals (DDC).

Cells were harvested at indicated time-points after exposure to genotoxic agents and protein levels were analyzed by immunoblot-

ting with specific antibody. DNA damage was confirmed by the phosphorylation of Ser 345 of Chk1 (top panel; P-Chk1). Open

arrows indicate cross reactive band while filled arrows point to specific band. LC (loading control) points to a nonspecific band

that displays equal protein load in different lanes. (Panel A and B, right panel) The immunoblots of different proteins were quanti-

fied and the numbers indicate levels after different treatments relative to control untreated cells. The levels of a particular protein

(Cdt1, Cdc6, Mcm10, Mcm6, Mcm7, TopBP1, PCNA, or POLe p59) after different treatments have been displayed adjacent to its

immunoblot. The standard deviation of this data has been reported in Supporting Information Table S1A.

767STRESS INDUCED PROTEOLYSIS OF REPLICATION FACTORS

that ionizing gamma radiation, UV radiation, DNA-damaging

chemicals, hydroxyurea, and hydrogen peroxide induced the

phosphorylation of p53, a known marker for DNA damage

(Supporting Information Figs. S1B and S3B) (56). We deter-

mined the levels of 23 replication proteins as their antibodies

worked efficiently in the immunoblotting assays and similarly

the intracellular localization was determined for 13 replication

proteins whose antibodies worked for immunofluorescence assay

(Fig. 3 and Supporting Information Fig. S2). The specificity of

the band observed in the immunoblotting assay or the fluores-

cence signal obtained in the immunofluorescence assay was

authenticated by observed mobility of SDS-PAGE gel and

RNAi against specific gene (Figs. 4 and 5; Supporting Informa-

tion Figs. S4A and S4B).

When HeLa and U2OS cells were exposed to UV, gamma

radiation, and genotoxic chemicals the stability of four subunits

of ORC was unaltered (Supporting Information Fig. S1) (57,

58). We observed that Cdc6, Cdt1, and Mcm10 were degraded

following UV-irradiation as has been previously reported (Figs.

2 and 3) (46, 48, 54). Although Cdt1 was decreased after expo-

sure to gamma radiation and DNA modifying chemicals, the

levels of Cdc6 and Mcm10 remained unchanged demonstrating

that mammalian cells target specific molecules based on the

type of stress experienced (Figs. 1 and 2). We wanted to deter-

mine the minimum dose required for the downregulation of

Cdt1, Cdc6, and Mcm10 and observed that Mcm10 and Cdc6

were downregulated after 50 J/m2 UV irradiation of HeLa cells

while level of Cdt1 was decreased with 25 J/m2 UV irradiation

(Fig. 4A). However, exposure of 25 Gy gamma radiation was

sufficient to downregulate the Cdt1 protein (Fig. 4A). In mam-

malian cells, inhibition of Cdc6 at G1/S boundary is due to its

export from the nucleus but no change in Cdc6 localization was

observed demonstrating that the natural mechanism of inhibiting

Cdc6 activity is not utilized during stress (Fig. 3). We did not

observe reduction in Cdc6 level by IF, probably because of pro-

teolysis into fragments, which are presumably smaller than the

range of gradient gel but detectable by high sensitivity of the

Cdc6 antibody in IF assays. Since the stability of MCM subu-

nits remained unaffected after exposure to different genotoxic

agents, we conclude that out of all the components of prerepli-

cation complex, Cdc6 and Cdt1 seem to be downregulated by

the cells for inhibiting replication (Figs. 2 and 3; Supporting In-

formation Figs. S2 and S3).

High CDK activity at the G1/S transition recruits the

Mcm10, Cdc45, TopBP1, DNA polymerase epsilon and GINS

complex to form the preinitiation complex followed by the RPA

complex (7, 8). We observed that TopBP1 was stable after ex-

posure to UV, gamma radiation and genotoxic chemicals (Fig.

2). A lower mobility, phosphorylated band of Rpa2 was

observed following UV and gamma irradiation (Figs. 2A and

Figure 3. Intracellular regulation of replication proteins in single cells assayed by immunofluorescence. HeLa (A) and U2OS (B)

cells grown on glass coverslips were exposed to 100 or 200 J/m2 UV (UV-rad), 50 Gy gamma radiation (c-rad) and DNA damag-

ing chemicals (DDC). Cells were harvested at indicated time-points after exposure to genotoxic agents, fixed with formaldehyde,

permeabilized with Triton X-100 followed by immunofluorescence with specific antibody and the localization was displayed with

secondary antibody conjugated to FITC (green color). DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) (blue color).

768 SHARMA ET AL.

2B) (59). Rpa2 forms a punctuate pattern 4 h post exposure to

gamma radiation and genotoxic chemicals, which is completely

absent in untreated HeLa cells (Fig. 3). After binding of RPA,

DNA polymerase alpha-primase is recruited to the unwound ori-

gins. We assayed the catalytic subunit of polymerase alpha,

p180, RFC subunits, and PCNA but did not observe any change

in their levels or localization in cells till 4 h after exposure to

genotoxic stress (Fig. 2; Supporting Information Figs. S1 and

S2). Similarly, the levels and localization of p17 and p59 subu-

nits of DNA polymerase epsilon remained unchanged following

DNA damage. The p50 subunit of polymerase delta was

stable after exposure to UV radiation unlike the p12 subunit,

which is known to be, destabilized (Supporting Information

Fig. S1) (60).

We wanted to identify the degradation pathway mediating

the downregulation of replication factors, Cdt1, Cdc6, and

Mcm10. We have previously shown that UV-triggered Mcm10

downregulation is not meditated by transcription control and ap-

optotic machinery (54). HeLa cells were treated with 50 lMMG-132, an inhibitor of the 26S proteasome, followed by UV

or gamma irradiation and harvested after 4 h (Fig. 4B). As

reported previously, MG132 prevented the downregulation of

Mcm10 after UV-irradiation. Downregulation of Cdc6 after

UV-irradiation was completely blocked in the presence of

MG132. We also observed that Cdt1 decrease after UV and

gamma-irradiation was suppressed by inhibiting the 26S protea-

some. Therefore, we conclude that 26S proteasome imparts a

vital function in stalling the replication under conditions of

stress by proteolyzing essential replication factors.

Regulation of Replication Apparatus on ReplicationStalling and Oxidative Stress

To impose replication stress on HeLa and U2OS cells, we

utilized hydroxyurea (HU), which inhibits the ribonucleotide re-

ductase preventing the reduction of ribonucleotides to deoxyri-

bonucleotides impeding the S phase progression. The activation

of checkpoint on treatment with HU was evident by phosphoryl-

ation of Chk1 (Figs. 5A and 5B, A0). Cdt1, Cdc6, and Mcm10

were downregulated by UV irradiation but remained stable after

Figure 4. Stress-triggered degradation of Cdt1, Cdc6, and Mcm10 is mediated by 26S proteasome. (A) Effect of different doses of

UV or gamma radiation on stability of replication proteins. HeLa cells were exposed to the indicated dose of UV or gamma radia-

tion (UV-rad, c-rad) and harvested after 4 h for analysis of protein levels by immunoblotting with specific antibody. (B) Regulation

of replication proteins following exposure to UV or gamma radiation in the presence of MG132. HeLa cells treated with 50 lMMG132 or plain DMSO were exposed to UV or gamma radiation (UV-rad, c-rad) and harvested 4 h later for analysis of protein

levels by immunoblotting. DNA damage was confirmed by the phosphorylation of Ser 345 of Chk1 (top panel; P-Chk1). LC

(loading control) points to RNase H1 band that displays equal protein load in different lanes. (Panel A, right panel) The immuno-

blots of different proteins were quantified and the numbers indicate levels after different treatments relative to control untreated

cells. The levels of a particular protein (Cdt1, Cdc6, Mcm10) after different treatments have been displayed adjacent to its immu-

noblot.

769STRESS INDUCED PROTEOLYSIS OF REPLICATION FACTORS

Figure 5. Regulation of replication apparatus on replication stalling and oxidative damage. A: HeLa (A) and U2OS (B) cells were

exposed to 1.3 mM hydroxyurea (HU), harvested at indicated time-points and protein levels were analyzed by immunoblotting with

specific antibody. Checkpoint activation was evidenced by phosphorylation of Ser 345 of Chk1 (top panel). B: HeLa (A) and

U2OS (B) cells were exposed to 2.5 mM hydrogen peroxide for 1 h, harvested 3 h later and protein levels were analyzed by immu-

noblotting with specific antibody. DNA damage was confirmed by the phosphorylation of Ser 345 of Chk1 (top panel). The levels

of replication proteins after exposure to different forms of stress were quantitated and the numbers in panel (A) and (B) indicate ra-

tio relative to control levels. The mean and standard deviation of two independent experiments have been reported. A(C0): Small in-

hibitory RNA was carried out against specific genes to confirm that the band obtained on immunoblotting is attributable to that rep-

lication protein. The observed mobility of the replication proteins was also in expectation with their calculated molecular mass:

Cdt1: 70 kDa; Cdc6: 63 kDa; Mcm10: 110 kDa; Mcm6: 110 kDa; Mcm7: 85 kDa; TopBP1: 180 kDa; PCNA: 38 kDa; Pol e p59:53 kDa; Rpa2: 32 kDa. LC (loading control) points to RNase H1 band that displays equal protein load in different lanes. (Panel A

and B, right panel) The immunoblots of different proteins were quantified and the numbers indicate levels after different treatments

relative to control untreated cells. The levels of a particular protein (Cdt1, Cdc6, Mcm10, Mcm6, Mcm7, TopBP1, PCNA, or POLep59) after different treatments have been displayed adjacent to its immunoblot. The standard deviation of this data has been

reported in Supporting Information Table S1B.

770 SHARMA ET AL.

replication stalling (Figs. 5 and 6, A0). Therefore, the replication

stress induced pathway does not utilize the downregulation of

these key replication molecules to inhibit replication. However,

replication arrest with hydroxyurea results in phosphorylation

and formation of punctuate pattern of Rpa2 reminiscent of

localization to the sites of replication arrest, which is clearly

absent in untreated U2OS cells (Figs. 5B and 6B, A0). The

levels and localization of other replication proteins remain unaf-

fected after hydroxyurea block (Supporting Information Figs. S3

and S4). In comparison to HeLa cells, the level of phosphoryla-

tion of Rpa2 was significantly higher in p53 positive U2OS

cells underlining that the stress response has to be addressed in

different genetic backgrounds (Figs. 5A and 5B, A0).Next, we wanted to study the effect of oxidative stress on

replication apparatus and therefore we exposed HeLa and U2OS

cells to 2.5 mM hydrogen peroxide (H2O2) for 1 h and cells

were harvested 3 h later. There was a time dependent increase in

reactive oxygen species and damage to DNA as indicated by

phosphorylation of Chk1 (Figs. 5A and 5B, B0). We observed

that the majority of replication proteins did not show any change

in levels or localization (Figs. 5 and 6, B0; Supporting Informa-

tion Figs. S3 and S4). We observed a partial decrease in the lev-

els of TopBP1 after oxidative stress to U2OS cells which was

not seen in HeLa cells (Figs. 5A and 6B, B0). HeLa cells are

infected with human papillomavirus whose products E7 and E6,

target the Rb and p53 protein and therefore these cells are defi-

cient in activities of these key checkpoint proteins (31, 32). How-

ever U2OS cell line which expresses wild type p53 and Rb par-

tially downregulates TopBP1 following oxidative stress (33). It is

well-known that p53 interacts with and activates many check-

point proteins and therefore it is likely that downregulation of

TopBP1 following DNA damage is mediated by a p53 dependent

mechanism signifying that targets for inhibiting replication may

vary in cells with different checkpoint activities (Figs. 5A and

5B, B0) (50, 52). We observed that Cdt1 was downregulated after

exposure to hydrogen peroxide indicating that it could be a possi-

ble target for inhibiting replication following oxidative stress

(Figs. 5A and 5B, B0). We also observed that Cdc6 levels were

partially reduced, suggesting its role in replication inhibition.

Mcm10, which was downregulated following UV irradiation did

not decrease, indicating that oxidative stress induced inhibition of

replication machinery is independent of Mcm10 (Figs. 5A and

Figure 6. Intracellular regulation of replication apparatus on replication stalling and oxidative damage. A: HeLa (A) and U2OS (B)

cells grown on glass coverslips were exposed to 1.3 mM hydroxyurea (HU), harvested at indicated time-points. Cells were har-

vested at indicated time-points after exposure to genotoxic agents, fixed with formaldehyde, permeabilized with Triton X-100 fol-

lowed by immunofluorescence with specific antibody and the localization was displayed with secondary antibody conjugated to

FITC (green color). DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) (blue color). B: HeLa (A) and U2OS (B) cells

grown on glass coverslips were exposed to 2.5 mM hydrogen peroxide for 1 h, harvested 3 h later, fixed with formaldehyde, per-

meabilized with Triton X-100 followed by immunofluorescence with specific antibody and the localization was displayed with sec-

ondary antibody conjugated to FITC (green color). DNA was stained with 4, 6-diamidino-2-phenylindole (DAPI) (blue color).

A(C0): To confirm that the signal obtained in the immunofluorescence assay is attributable to the specific replication protein, RNAi

mediated depletion of specific genes was carried out followed by immunofluorescence with corresponding antibody.

771STRESS INDUCED PROTEOLYSIS OF REPLICATION FACTORS

5B, B0). We observed that TopBP1 is stable after exposure to

UV, gamma irradiation and genotoxic chemicals in U2OS cells

but seems to be a target of oxidative stress induced damage,

highlighting that different genotoxic agents target specific factors

for inhibiting replication (Fig. 5B, B0).

DISCUSSION

On experiencing DNA damage, protective checkpoint path-

ways are invoked to stall the ongoing DNA replication. Defi-

ciency in cell cycle checkpoint regulation that results in radiore-

sistant DNA synthesis after irradiation leads to chromosomal

instability (61). It is well accepted that the regulation of the ac-

tivity of DNA replication genes is critical for maintaining

genomic integrity of the cell and deregulation results in various

forms of genomic instability (62–65). Since deregulation of rep-

lication genes will inadvertently lead to genomic lesions, it is

logical to study the downregulation of replication genes that is

believed to arrest the replication process. In this study, we have

attempted to establish the stability of protein products of wide

range of DNA replication genes. Previous attempts have eval-

uated the mRNA levels by microarray and identified many cell-

cycle and replication genes that are transcriptionally downregu-

lated after DNA damage (28–30). However, this approach was

unable to identify changes in replication proteins that are likely

to contribute to immediate cessation of DNA replication. Inci-

dental discoveries have identified some replication genes that

are downregulated following stress but there has been no sys-

tematic study that addressed the regulation of replication ma-

chinery after different forms of DNA damage. There has been

constant speculation about the replication factors that are ‘‘regu-

lated’’ during stress and it has been debated that many factors

would be downregulated for stalling ongoing replication. For

the first time, we describe the stability of the entire replication

apparatus following DNA damage by individually evaluating

the levels and localization of entire spectrum of the replication

proteins. Our study conclusively identifies the effect of different

stress on individual replication factors. Summing up, our study

makes the following findings.

Following DNA damage, the regulatory machinery targets

key replication factors: All the four replication factors targeted

following DNA damage are essential for the replication process.

Cdc6 is an ATPase that binds to the origins after origin recogni-

tion complex during the M/G1 phase, which is relatively an

early stage of replication. Since it is essential for loading MCM

proteins onto the DNA, its downregulation would inadvertently

prevent replication initiation (66). Replication initiator Cdt1

functions along with Cdc6 to form the prereplicative complex

(pre-RC) (67, 68). It is an essential DNA licensing factor whose

activity is regulated in a cell-cycle manner (69, 70). In higher

eukaryotes, Mcm10 associates with the replication machinery at

a later stage of DNA replication: Bound MCM helicase is

essential for the loading of Mcm10 protein to the origins of rep-

lication. Mcm10 is essential for the association of DNA poly-

merase alpha p180 with chromatin and its depletion blocks S

phase progression, demonstrating its essential requirement in

elongation (71). Therefore, Mcm10 is essential for both the ini-

tiation and elongation phases of replication and decrease in its

levels after DNA damage will stall ongoing replication. p12

subunit is an essential component of the DNA polymerase delta

and consequently its loss compromises the activity of the poly-

merase that is essential for DNA replication elongation. There-

fore the regulatory machinery targets replication factors that

function at different stages of replication, apparently employing

a guarded mechanism to inhibit replication.

In this study, we imposed different kinds of stress to discern

whether similar or unique responses are invoked by mammalian

cells. Cdt1 is downregulated after UV, gamma irradiation, and

oxidative stress whereas Cdc6 and Mcm10 are degraded on ex-

posure to UV radiation (46, 48). Although our results demon-

strate that specific replication factors are targeted based on the

genotoxic agent, and the downregulation of different replication

factors utilizes a similar pathway: ubiquitination and degrada-

tion by the 26S proteasome (Fig. 4A). Although degradation by

26S proteasome is a common step in stress-induced downregu-

lation of replication proteins, independent ubiquitin ligases are

utilized to ensure redundancy in responding to stress. Cdc6

ubiquitination is mediated by HECT domain E3 ligase,

HUWE1, whereas Cdt1 utilizes an E3 ligase comprising of a

cullin, Cul4, a ring finger protein, Roc1, adaptor protein,

DDB1, and a substrate-recognition subunit, Cdt2 (65, 72–75).

Mcm10 downregulation utilizes Cul4-Roc1-DDB1 complex but

may be independent of Cdt2, thereby preserving independent

regulation of Mcm10 levels following DNA damage (54).

Our study did not focus on the post-translational modifica-

tions like ubiquitination, sumoylation, and phosphorylation that

are apparently involved in regulating activity of key replication

proteins. However, to determine modifications of replication

proteins in the entire mammalian cell proteome remains a major

challenge for mass spectrometry techniques and it will require

significant advancement of tools before this information would

be available. This probably explains no reported attempt to

understand the stress-induced modifications in replication appa-

ratus as a whole. It is important to note that degradation is also

an established means by which human cells bring about imme-

diate cessation of DNA replication. Kondo et al observed that

the inhibition of DNA synthesis after UV treatment of HeLa

cells is interfered by ectopic expression of Cdt1 (76). This

result clearly demonstrates that the degradation of Cdt1 is im-

portant for the inhibition of DNA synthesis following DNA

damage. We observed that following UV irradiation, BrdU

incorporation was higher in Mcm10-overexpressing cells than in

non-Mcm10-overexpressing cells signifying that Mcm10 is a

limiting factor whose activity regulates DNA replication follow-

ing stress (54). Therefore, this study addresses one of the means

by which stress-induced regulation of replication is achieved.

U2OS and HeLa cells are functionally different in p53 and

Rb activity and have been utilized by many groups for under-

772 SHARMA ET AL.

standing the regulation of replication apparatus following stress

(46–48). While comparing these cell lines with different back-

grounds, we did not observe major differences in response to

stress: Downregulation of key proteins, Cdc6, Cdt1, and

Mcm10, was similar in these cell lines. However, we have

observed that the PI3K-related kinases, ATM/ATR are required

for downregulation of Mcm10 during stress. Therefore, the role

of the checkpoint pathways in regulating replication machinery

during stress would be better understood by comparing cell

lines mutated in key checkpoint proteins. In conclusion, our

study demonstrates that on exposure to stress, only key replica-

tion factors essential for formation of prereplication and preini-

tiation complexes are downregulated to inhibit progressing rep-

lication. We also demonstrate that different forms of stresses

may share the same replication target or may inhibit unique

targets for stalling replication. We propose a model for inhibi-

tion of replication machinery in which mammalian cells

target specific essential replication factors based on the stress

experienced.

ACKNOWLEDGEMENTS

We thank Mahendran Chinnappan, Vinayak Khattar, Chetan

Jain, Md. Muntaz Khan, Kanika Jain, and Sneha Shah in assist-

ing in IB and IF assays and Puneeta Bhatia for her help in proc-

essing the images. A.S., A.K., M.K. carried out the major

assays with assistance from S.A., S.M., K.R., and S.R. This

work was funded by Department of Biotechnology, Government

of India, Grant Number: BT/PR10835/NNT/28/116/2008.

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