Report

PTEN Controls the DNA Re

plication Process throughMCM2 in Response to Replicative Stress

Graphical Abstract

Highlights

d PTEN regulates DNA replication through MCM2

d PTEN dephosphorylates MCM2 in response to replication

stress

d Dephosphorylation of MCM2 restricts replication fork

progression

d PTEN prevents fork progression upon fork stalling and

maintains genomic stability

Feng et al., 2015, Cell Reports 13, 1295–1303November 17, 2015 ª2015 The Authorshttp://dx.doi.org/10.1016/j.celrep.2015.10.016

Authors

Jiawen Feng, Jing Liang, Jiaju Li, ...,

Xuyang Zhao, Michael A. McNutt,

Yuxin Yin

Correspondenceyinyuxin@hsc.pku.edu.cn

In Brief

Feng et al. report a mechanism by which

nuclear PTEN functions to protect the

genome. PTEN controls replication fork

progression through MCM2. Upon

replicative stress, PTEN

dephosphorylatesMCM2 at serine 41 and

restricts fork progression, thus

maintaining genome stability.

Cell Reports

Report

PTEN Controls the DNA Replication Processthrough MCM2 in Response to Replicative StressJiawen Feng,1,2 Jing Liang,1,2 Jiaju Li,1 Yunqiao Li,1 Hui Liang,1 Xuyang Zhao,1 Michael A. McNutt,1 and Yuxin Yin1,*1Institute of Systems Biomedicine, Center for Molecular and Translational Medicine, Department of Pathology, School of Basic Medicine,Peking-Tsinghua Center for Life Sciences, Peking University Health Science Center, Beijing 100191, PRC2Co-first author

*Correspondence: yinyuxin@hsc.pku.edu.cn

http://dx.doi.org/10.1016/j.celrep.2015.10.016This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

SUMMARY

PTEN is a tumor suppressor frequently mutated inhuman cancers. PTEN inhibits the phosphatidylino-sitol 3-kinase (PI3K)-AKT cascade, and nuclearPTEN guards the genome by multiple mechanisms.Here, we report that PTEN physically associateswith the minichromosome maintenance complexcomponent 2 (MCM2), which is essential for DNAreplication. Specifically, PTEN dephosphorylatesMCM2 at serine 41 (S41) and restricts replicationfork progression under replicative stress. PTENdisruption results in unrestrained fork progressionupon replication stalling, which is similar to thephenotype of cells expressing the phosphomimicMCM2 mutant S41D. Moreover, PTEN is necessaryfor prevention of chromosomal aberrations underreplication stress. This study demonstrates thatPTEN regulates DNA replication through MCM2and loss of PTEN function leads to replication de-fects and genomic instability. We propose thatPTEN plays a critical role in maintaining genetic sta-bility through a replication-specific mechanism, andthis is a crucial facet of PTEN tumor suppressoractivity.

INTRODUCTION

Germline and somatic PTEN mutations are found frequently in

human cancer (Chalhoub and Baker, 2009; Hobert and Eng,

2009). Despite the important role PTEN plays in antagonizing

the PI3K-AKT pathway in the cytoplasm (Chen et al., 2006;

Stambolic et al., 1998), emerging evidence argues that nuclear

PTEN participates in multiple anti-cancer processes and, in

particular, participates in maintenance of genomic integrity to

suppress tumorigenesis (Bassi et al., 2013; Chen et al., 2014;

Shen et al., 2007; Song et al., 2011; Sun et al., 2014). PTEN defi-

ciency is associated with common fragile sites instability (Sun

et al., 2014), and fragile sites instability is a common feature of

DNA replication dysfunction (Franchitto and Pichierri, 2014; Ma-

cheret and Halazonetis, 2015). Recent evidence from mass

Cell Rep

spectrometry studies also suggests that PTEN may associate

with replication fork components (Alabert et al., 2014), and these

findings suggest that PTEN may be involved in replication-

related function.

Throughout the cell cycle, under normal conditions, DNA is

most prone to breakage during S phase (Aguilera and Go-

mez-Gonzalez, 2008). DNA replication must be closely coordi-

nated with cell cycle checkpoints, DNA repair, and other

cellular processes to ensure chromosome maintenance (Bran-

zei and Foiani, 2010). Replication stress caused by replication

fork barriers, nucleotide deprivation, or defects of replication-

associated enzymes leads to genomic instability and must be

counteracted (Aguilera and Gomez-Gonzalez, 2008; Kolodner

et al., 2002; Labib and Hodgson, 2007; Schlacher et al.,

2011). The replication fork stalls when it suffers replication

stress. Early studies suggested that S phase checkpoint ki-

nases prevent replisome disassembly at the stalled replication

folk (Heller and Marians, 2006; Lucca et al., 2004). However,

recent direct evidence has shown that the replisome is unex-

pectedly stable, even in cells with a deficiency of checkpoint

kinase under replication stress (De Piccoli et al., 2012), and un-

restrained replication fork progression results in fork collapse

on replication stalling (Lossaint et al., 2013; Sabatinos et al.,

2012), suggesting that restraint of fork progression is necessary

for the replication stress response. The MCM2-7 complex is

one of the core components of the replisome (Bochman and

Schwacha, 2008; Labib et al., 2000) and plays crucial roles in

replication origin firing, elongation, termination, and replication

stress response (Blow and Dutta, 2005; Lossaint et al., 2013;

Maric et al., 2014; Moreno et al., 2014; Tenca et al., 2007).

Recent studies have shown that the MCM2-7 complex is

involved in inhibition of fork progression upon replication stall-

ing (Lossaint et al., 2013; Sabatinos et al., 2012; Stead et al.,

2011). In this study, we demonstrate that PTEN dephosphory-

lates MCM2 and inhibits replication fork progression under

replication stress, suggesting that PTEN is involved in an S

phase checkpoint. DNA molecular combing analysis revealed

that replication forks in PTEN-proficient cells are subject to

more efficient braking in response to fork stalling than the

forks in PTEN-deficient cells. Moreover, we found that PTEN-

proficient cells exhibit decreased chromosomal aberrations

under replication stress. Our findings indicate that PTEN acts

as guardian of the genome through a replication-specific

mechanism.

orts 13, 1295–1303, November 17, 2015 ª2015 The Authors 1295

Figure 1. MCM2 Is a PTEN-Associated Protein that Acts in Response to Replication Stress

(A) Re-expression of exogenous S-tag-PTEN in HepG2 PTEN knockdown cells. The left panel illustrates the knockdown efficiency (lane 2 versus lane 1), and the

right panel shows the expression level of exogenous PTEN (lane 4 versus lane 3). A small hairpin RNA was designed to target the 50 UTR region of PTEN mRNA.

This shRNA thus diminishes only endogenous and not exogenous PTEN expression.

(B) Mass spectrum analysis showed that MCM2 is a potential PTEN-binding partner. Sequences of peptides derived from MCM2 identified by mass spectrum

analysis are shown.

(C) PTEN associates with MCM2 upon fork stalling. PTEN+/+ and PTEN�/� cells were treated with 4 mM hydroxyurea (HU) or DMSO for 2 hr. PTEN and its

associated proteins immunoprecipitated with a PTEN mouse monoclonal antibody (sc-7974) from cell extracts were subjected to western blotting using an

MCM2 antibody generated in our laboratory and a PTEN rabbit polyclonal antibody (rabbit; CST no. 9559). In the input panel, MCM2 and PTEN expression levels

were analyzed and GAPDH served as loading control.

(D) Amoderate degree of PTEN-MCM2 interaction is detectable under unperturbed conditions. After treatment with 4mMHU (perturbed) or DMSO (unperturbed)

for 2 hr, PTEN�/�U2OS cells expressing control S-tag or S-tag-PTEN were lysed, followed by S-protein pull-down and western blotting with MCM2 antibody and

PTEN antibody (sc-7974). In the input panel, MCM2 and PTEN expression levels were analyzed and GAPDH served as loading control.

RESULTS

PTEN Associates withMCM2 in Response to ReplicationStressOur previous studies indicate that PTEN deficiency results in

instability of replication-associated common fragile sites (Sun

et al., 2014), which led us to further investigate the relationship

of PTEN and replication stress. To determine whether PTEN is

involved in the replication stress response, we isolated PTEN-

associated complexes from cells treated with hydroxyurea,

which restrains DNA replication by depletion of dNTP pools

1296 Cell Reports 13, 1295–1303, November 17, 2015 ª2015 The Au

(Koc et al., 2004). To reduce the competing effect of endogenous

PTEN, S-tag (S-protein-binding peptide) or S-tag-PTEN was re-

expressed in HepG2 cells where PTEN expression wasmarkedly

reduced by shRNA (Figure 1A). S-tag pull-down followed by

mass spectrum analysis revealed that MCM2, an important

regulator of DNA replication, is a potential PTEN-associated pro-

tein (Figure 1B). Results of immunoprecipitation carried out with

U2OS cells and HCT116 cells revealed that interaction of endog-

enous PTEN and MCM2 occurs in response to hydroxyurea

treatment (Figure 1C, lane 4 versus lanes 1–3; lane 8 versus lanes

5–7; somatic knockout cell lines are described in Figure S1),

thors

suggesting that this interaction is a part of the replication stress

response. We noticed that dominant expression of exogenous

PTEN leads to a moderate degree of PTEN-MCM2 interaction

under physiological conditions (Figure 1D, lane 2), and this inter-

action was strengthened by hydroxyurea treatment (Figure 1D,

lane 4), suggesting that PTEN may counteract even physiologic

replication stress through this mechanism.

PTEN Dephosphorylates MCM2 at S41 upon ReplicationFork StallingMCM2 is a key regulatory component of the MCM2-7 complex,

which is a core replication helicase of the replisome (Bochman

and Schwacha, 2008), and its function is modulated by MCM2

phosphorylation status (Bruck and Kaplan, 2009; Charych

et al., 2008; Chuang et al., 2009; Montagnoli et al., 2006; Tenca

et al., 2007; Tsuji et al., 2006). PTEN is a well-known protein

phosphatase (Gu et al., 2011; Li et al., 1997; Shi et al., 2014),

and it is therefore possible that PTEN modulates the MCM2

phosphorylation status. To test this possibility, we introduced

S-tag-MCM2 into HCT116 PTEN+/+ and PTEN�/� cells, followed

by treatment of these cells with hydroxyurea and subsequent

purification of the exogenous MCM2 with S-protein beads

(Figure 2A). Mass spectrum analysis demonstrated that the

S41-phosphorylated peptide intensity was 4-fold greater in

PTEN�/� cells than that in PTEN+/+ cells, whereas the phosphor-

ylated peptide intensities of S27, S139, and S108 differed little in

these two cell types (Figures 2B and S2). In vitro dephosphoryla-

tion assays indicated that PTEN dephosphorylatesMCM2 at S41

(Figures 2C, lane 2, and 2D, lanes 4–6 versus lanes 1–3). As

shown in Figures 2E–2G (lane 4 versus lane 3), this increase in

phosphorylated MCM2 at S41 was observed upon replication

stalling in PTEN�/� MEFs and PTEN-deficient U2OS and

HCT116 cells. Under replication stress, the level of phosphory-

latedMCM2 (S41) was consistently increased in cells expressing

PTEN C124S, which lacks both protein- and lipid-phosphatase

activity, as compared with cells expressing wild-type PTEN or

PTEN G129E, which retains its protein-phosphatase activity

(Figure 2H, lane 7 versus lane 6 and lane 8). These data indicate

that PTEN is involved in regulation of the MCM2 phosphorylation

status.

MCM2Dephosphorylation at S41 Leads to Restriction ofFork ProgressionThe relationship of PTEN and MCM2 phosphorylation status

raised a question as to the role of MCM2 S41 phosphorylation

in response to replication stress. Phosphorylation of MCM2 at

different sites results in differing physiologic functions, including

pre-replication complex assembly, modulation of fork progres-

sion, and replication origin firing (Chuang et al., 2009;Montagnoli

et al., 2006; Stead et al., 2011; Tenca et al., 2007; Tsuji et al.,

2006). However, the role of MCM2 S41 phosphorylation has

not beenwell established. Emerging evidence has demonstrated

that efficient replication fork pause is essential for preventing sin-

gle-stranded DNA accumulation and subsequent fork collapse in

stressful situations (Lossaint et al., 2013; Sabatinos et al., 2012).

Interestingly, we found that PTEN is also associated with other

components of the MCM complex (Figure S3), suggesting that

PTEN may influence the function of the MCM complex.

Cell Rep

In view of the importance of restraint of fork progression upon

replication stalling and the role of MCM proteins (Lossaint et al.,

2013; Stead et al., 2011), we sought to determinewhetherMCM2

S41 phosphorylation is involved in fork progression. Using repli-

cation analysis of single DNA molecules by molecular combing,

we evaluated the involvement of MCM2 S41 phosphorylation in

fork progression. U2OS cells expressing control S-tag, wild-type

MCM2, phospho-dead MCM2 S41A, or phosphomimic MCM2

S41D (Figure 3A) were double labeled, first with CldU for

20 min and then with IdU for another 20 min (Figure 3B, upper

panel). As shown in Figure 3B, lower panel, IdU tracts adjacent

to CldU tracts in cells expressing MCM2 S41D were significantly

longer than those in other groups, reflecting a higher rate of repli-

cation fork progression. In contrast, IdU tracts in cells expressing

MCM2 S41A were significantly shorter (Figure 3B, lower panel).

These cells were also labeled with CldU for 20 min, followed

by IdU labeling and simultaneous treatment with hydroxyurea

for 2 hr (Figure 3C, upper panel). IdU tracts in cells expressing

MCM2 S41A were consistently significantly shorter than those

in other groups, whereas IdU tracts in cells expressing MCM2

S41D were significantly longer (Figure 3C, lower panel). These

observations suggest that dephosphorylation of MCM2 S41 is

involved in restriction of fork progression. To address the role

of PTEN in modulation of fork progression upon fork stalling,

PTEN+/+ and PTEN�/� U2OS cells were labeled first with CldU

and then labeled with IdU and simultaneously treated with hy-

droxyurea as described above. IdU tracts in PTEN�/� cells

were significantly longer than those in PTEN+/+ cells (Figure 3D,

PTEN�/�HU versus PTEN+/+ HU), similar to the IdU tracts in cells

expressing MCM2 S41D (Figure 3C, MCM2-S41D HU). More-

over, we confirmed that PTEN has only minimal impact on the

length of IdU tracts under unperturbed conditions (Figure 3D,

PTEN+/+ versus PTEN�/�). A well-known PI3K inhibitor (Gharbi

et al., 2007; Hu et al., 2000), LY294002 was also used to

determine the relevance of the PI3K-AKT pathway in replication

fork progression. Despite the significantly decreased levels of

pAKT (S473) in LY294002-treated PTEN�/� cells, LY294002 fails

to suppress MCM2 (S41) phosphorylation (Figure 3E, lane 3

versus lane 2; lane 6 versus lane 5). The length of IdU tracts

in LY294002-treated PTEN�/� cells was still consistently sig-

nificantly longer than that in PTEN+/+ cells upon fork stalling

(Figure 3D, PTEN+/+ HU versus PTEN�/� HU+ LY294002;

p < 0.0001), consistent with the higher level ofMCM2 (S41) phos-

phorylation in LY294002-treated PTEN�/� cells (Figure 3E, lane 6

versus lane 4). These results suggest that PTEN may modulate

fork rate through its influence on the MCM2 phosphorylation

status.

PTEN Counteracts Replication Stress and MaintainsGenome StabilityDNA fiber combing analysis (as described in Figure 3C) showed

that cells expressing PTEN C124S exhibit continued fork pro-

gression (Figure 4A, PTEN-C124S versus PTEN-WT and PTEN-

G129E), consistent with the phosphorylation levels ofMCM2S41

shown in Figure 2H. In addition, ectopic expression of theMCM2

S41A mutant, but not wild-type MCM2 or the MCM2 S41D

mutant, compromises the increased fork rate in PTEN�/� cells.

Re-expression of wild-type PTEN, PTEN G129E, or MCM2

orts 13, 1295–1303, November 17, 2015 ª2015 The Authors 1297

Figure 2. MCM2 Is a Protein Substrate of PTEN Phosphatase

(A) Purification of MCM2 protein in PTEN+/+ and PTEN�/� cells. PTEN+/+ or PTEN�/� HCT116 cells transfected with S-tag-MCM2 were treated with 4 mM HU for

2 hr and then extracted in lysis buffer containing phosphatase inhibitors. Purified S-tag MCM2 proteins were subjected to SDS-PAGE and stained with

Coomassie Blue.

(B) Peptide intensities of phosphorylated MCM2 peptides in PTEN+/+ or PTEN�/� HCT116 cells are shown in the table.

(C and D) PTEN dephosphorylates MCM2 at S41 in vitro. One microgram of GST, GST-PTEN, or GST-PTEN 124S protein was incubated with 1 mg His-MCM2

purified from SF9 cells in dephosphorylation buffer for 30 min (C). Dose dependency was also tested. One microgram of MCM2 was incubated with different

amounts of GST (2 mg, 1 mg, or 0.5 mg) or GST-PTEN (2 mg, 1 mg, or 0.5 mg) protein in dephosphorylation buffer for 30 min (D). GST, GST-PTEN, and GST-PTEN

124S were immunoblotted with a GST antibody. Both S41-phosphorylated MCM2 and total MCM2 were analyzed.

(E–G) Evaluation of MCM2 pS41 levels by western blotting in PTEN+/+ and PTEN�/� cells treated with 4 mM HU or DMSO for 2 hr. Total MCM2 expression levels

were analyzed, and GAPDH served as a loading control.

(H) PTEN�/� U2OS cells expressing control S-tag, PTEN, PTEN C124S, or PTEN G129E were treated with 4 mM HU (perturbed) or DMSO (unperturbed) for 2 hr

and then extracted in lysis buffer with phosphatase inhibitors. MCM2 pS41 levels were evaluated with western blotting. The levels of total MCM2 expression were

analyzed.

1298 Cell Reports 13, 1295–1303, November 17, 2015 ª2015 The Authors

Figure 3. MCM2 S41 Phosphorylation Mod-

ulates Replication Fork Progression

(A) Ectopic expression of wild-type MCM2 or

MCM2mutants in U2OS cells. Expression levels of

exogenous MCM2 were evaluated by western

blotting with an HA antibody.

(B) MCM2 phosphorylation status influences fork

progression in unperturbed conditions. Indicated

cells were labeled with CldU for 20 min and then

with IdU for another 20 min. Lengths (mm) of IdU

tracks adjacent to CldU tracks were measured to

evaluate fork progression.

(C) MCM2 phosphorylation status influences fork

progression in perturbed conditions. To evaluate

fork progression under replication stress, indi-

cated cells were double labeled, first with CldU for

20 min and then with IdU and simultaneously

treated with 0.5 mM HU for 2 hr. Lengths (mm) of

IdU tracks adjacent to the CldU tracks were

measured.

(D) Unrestrained fork progression in PTEN-defi-

cient cells upon fork stalling. PTEN+/+, PTEN�/�,and LY294002-treated PTEN�/� cells were

labeled as described in (B) and (C), and lengths

(mm) of IdU tracks adjacent to the CldU tracks

were measured. In (B)–(D), more than 120 tracts

were measured in each group, and crossbars

represent median tract lengths. The Mann-Whit-

ney rank sum two-tailed test was used to show

statistical differences. ns, no significant differ-

ence; ****p < 0.0001.

(E) Levels of pMCM2 (S41) are not attenuated by

LY294002. Lysates from PTEN+/+, PTEN�/�, andLY294002-treated PTEN�/� cells were subjected

to immunoblotting with indicated antibodies.

S41A, but not of PTENC124S, wild-typeMCM2, orMCM2S41D,

prevents replication stress-induced chromosomal aberrations

(Figure 4B). Moreover, reintroduction of wild-type PTEN in

U2OS PTEN�/� cells results in decreased accumulation of

chromosomal aberrations under both unperturbed and per-

turbed conditions (Figures 4C and 4D), suggesting that PTEN

counteracts even physiologic replication stress tomaintain chro-

mosomal integrity, which is consistent with Figure 1D (lane 2).

Interestingly, cells with ectopic expression of MCM2 S41A or

the S41D mutant exhibit more chromosomal aberrations as

compared with cells with wild-type MCM2 expression in unper-

turbed conditions (Figure 4C), whereas cells expressing MCM2

S41D exhibit increased accumulation of chromosomal aberra-

tions in perturbed conditions (Figure 4D), suggesting that the

phosphorylation status of MCM2 S41 must be tightly regulated

to prevent genomic instability.

DISCUSSION

Previous work has demonstrated that PTEN plays an essential

role in the maintenance of genome stability (Puc et al., 2005;

Cell Reports 13, 1295–1303, No

Shen et al., 2007; Song et al., 2011; Sun

et al., 2014). However, it is unclear how

PTEN functions to achieve this goal.

DNA replication is a crucial stage of the

cell cycle, and protection of the DNA replication process under

stress is necessary to ensure fidelity of replication and the

genome stability. In this paper, we report that PTEN controls

replication fork progression and PTEN does so through dephos-

phorylation of MCM2 under replication stress and thus guards

the genome.

MCM2 is an essential regulatory subunit of the MCM2-7 com-

plex that is loaded onto DNA during G1 phase and subsequently

activated by cyclin-dependent kinase (CDK) and Dbf4-depen-

dent kinases (DDKs), thus triggering DNA replication origin

licensing and firing (Labib, 2010). MCM2 deficiency in mice re-

sults in genome instability and cancer predisposition (Kunnev

et al., 2010; Pruitt et al., 2007; Rusiniak et al., 2012). MCM2 is

overexpressed in multiple types in cancers, which predicts

poor prognosis (Liu et al., 2013; Obermann et al., 2005; Wharton

et al., 2001; Yang et al., 2012). These observations argue that

MCM2 should be tightly controlled to maintain homeostasis of

DNA metabolism and chromosomal integrity. Because PTEN is

physically associated with MCM2 and dephosphorylates

MCM2, PTEN may influence functions of the MCM complex

through modification of MCM2 protein.

vember 17, 2015 ª2015 The Authors 1299

Figure 4. PTEN Prevents Chromosomal Instability by Resolving Replication Stress

(A) Re-expression of PTEN or MCM2 S41A in PTEN�/� cells rescues unrestrained fork progression. Lengths of IdU tracks adjacent to CldU tracks in PTEN�/�

U2OS cells expressing control S-tag, PTEN, PTENC124S, PTENG129E,MCM2,MCM2S41A, orMCM2 S41D labeled as described in Figure 3Cweremeasured.

More than 120 tracts weremeasured in each group, and crossbars representmedian tract lengths. TheMann-Whitney rank sum two-tailed test was used to show

statistical differences. ****p < 0.0001.

(B) PTEN and MCM2 S41A prevent HU-induced chromosomal aberrations. PTEN�/� U2OS cells expressing control S-tag, PTEN, PTEN C124S, PTEN G129E,

MCM2, MCM2 S41A, or MCM2 S41D treated with HU (perturbed) or DMSO (unperturbed) for 7 hr were subjected to metaphase spread analysis. Chromosomal

aberrations observed included gaps (red arrowhead), breaks (blue arrowhead), and dicentric chromosomes (green arrowhead). Crossbars represent the

mean number of chromosomal aberrations per cell. Data were statistically analyzed with the two-tailed Student’s t test. Error bars indicate SEM (n = 50). *p < 0.05;

**p < 0.01.

(C and D) Data derived from (B) were analyzed by separation into unperturbed (C) and HU-perturbed (D) groups. Crossbars represent the mean number

of chromosomal aberrations per cell. Data were statistically analyzed with the two-tailed Student’s t test. Error bars indicate SEM (n = 50). *p < 0.05; **p < 0.01;

***p < 0.001.

(E) Schematic model. In response to replication stress, PTEN associates with MCM2 and mediates MCM2 dephosphorylation at S41, which restricts fork

progression and results in pause of replication forks.

1300 Cell Reports 13, 1295–1303, November 17, 2015 ª2015 The Authors

Replication stress induces the PTEN-MCM2 interaction, sug-

gesting that PTEN functions to protect the DNA replication pro-

cess against the stress. Our mass spectrum analysis revealed

that phosphorylation of MCM2 at S41 is impaired by PTEN.

In vitro dephosphorylation analysis further confirmed that

PTEN is a phosphatase for MCM2 at S41 residue. However,

the exact effect of MCM2 S41 phosphorylation was still unclear.

Phosphorylation of MCM2 has been reported to be involved in

multiple functions in S phase (Bruck and Kaplan, 2009, 2015;

Chuang et al., 2009; Montagnoli et al., 2006; Stead et al., 2011;

Tenca et al., 2007; Tsuji et al., 2006). CDC7 phosphorylates

MCM2 at S27/41/139 during replication initiation (Tsuji et al.,

2006). However, more-detailed study argued that MCM2 S41

was phosphorylated by CDK2, but not CDC7 (Montagnoli

et al., 2006). Further investigations indicate that MCM2 was

phosphorylated at S40/53/108 by CDC7, improving cell viability

upon replication stress (Tenca et al., 2007). However, there is no

report of any phosphatase for MCM2 thus far. Our study demon-

strates that PTEN is a phosphatase for MCM2 and that this

dephosphorylation of MCM2 at S41 is involved in control of the

replication process upon replication stress.

It has been shown that unrestrained DNA synthesis acceler-

ates fork breakage upon replication stress (Lossaint et al.,

2013; Sabatinos et al., 2012). A recent report demonstrates

that PTEN facilitates loading of RAD51 onto DNA and regulates

the replication process (He et al., 2015). These observations

and our data suggest that PTEN controls replication fork pro-

gression to prevent DNA damage catastrophe during replication

stress through multiple mechanisms. It will be important to

further determine whether PTEN regulates other aspects of the

replication process.

Taken together, our findings provide evidence that PTEN is

involved in restriction of the fork rate through regulation of

MCM2 in the response to replication stress. We propose that

PTEN opposes the challenge of replication stress through its

multiple targets in order to prevent genomic instability. Our study

extends the understanding of PTEN’s role in maintaining

genomic integrity and sheds lights on how PTEN achieves multi-

farious tumor suppressor activity through participation in various

cellular processes.

EXPERIMENTAL PROCEDURES

DNA Fiber Assay

Cells were grown in 60-mmdishes to�70%confluence. Elongating replication

forks were labeled with CldU (50 mM) for 20 min. Before labeling with IdU

(50mM), cells were washed three times with PBS. The procedure for DNA fiber

spreads has been described previously (Jackson and Pombo, 1998). Briefly,

cells were harvested with trypsin digestion and resuspended in cold PBS.

Cell suspension was spotted onto one end of a microscope slide and mixed

at 1:6 with lysis buffer (0.5% SDS, 50 mM EDTA, and 200 mM Tris-HCl).

This mixture was incubated at room temperature for 4 min, and the slide

was then tilted at a 15� angle to allow spreading of DNA fibers down the glass

surface. DNA fibers were then fixed with 75% methanol and 25% acetic acid,

followed by acid denaturation with HCl (2.5M). Slideswere washed three times

with PBS before blocking with 5%BSA in PBS overnight at 4�C or 1 hr at 37�C.CldU- and IdU-labeled DNA fibers were analyzed by incubation with rat anti-

BrdU antibody (1:200 in blocking buffer; Novus Biologicals; BU1/75 [ICR1])

and mouse anti-BrdU antibody (1:100 in blocking buffer; Biosciences; clone

B44) for 2 hr at 37�C, followed by incubation with secondary antibodies

Cell Rep

(both diluted 1:200 in blocking buffer; Invitrogen; Alexa 488 goat anti-rat and

Alexa Fluor 555 goat anti-mouse) for 1 hr at room temperature. Slides were

mounted prior to observation using an Olympus IX51 microscope. Fibers

were analyzed using Cellsens Standard software (Olympus). Prism software

was used for statistical analysis. IdU and CldU reagents were purchased

from Sigma.

Karyotic Analysis

Cells were swollen with 0.075MKCl (typically for 20min at 37�C) and fixedwith

methanol/acetic acid (3:1) for 10 min. This fixation step was repeated three

times, and cells were resuspended with 1 ml fixation buffer, then dropped

on slides and stained with 5% Giemsa, and mounted on slides. Images were

obtained with an Olympus IX51 microscope.

Statistical Methods

For DNA molecular combing analysis, more than 120 tracts were measured in

each group and the Mann-Whitney rank sum two-tailed test was used to show

statistical differences. For karyotic analysis, 50 spreads were measured in

each group and data were statistically analyzed with the two-tailed Student’s

t test. p values less than 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****) were

considered statistically significant.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures

and three figures and can be found with this article online at http://dx.doi.

org/10.1016/j.celrep.2015.10.016.

AUTHOR CONTRIBUTIONS

Y.Y. and J.F. conceived the project and designed the study. J.F., J. Liang, J. Li,

Y.L., and H.L. performed experiments. X.Z. performedmass spectrometry and

analyzed MS data. Y.Y., J.F., and J. Liang analyzed data. The manuscript was

written by Y.Y. and J.F. and edited by J. Liang and M.A.M.

ACKNOWLEDGMENTS

We thank Wenhui Zhao, Yu Liu, Guangxi Wang, Xi Chen, Yang Li, Yu Zhang,

Danhui Ruan, and other members of the laboratory for technical assistance

and critical discussion. This study was supported by the National Natural

Science Foundation of China (81430056, 31420103905, 81372491, and

81321003), the 111 Project (B07001), the Shu Fan Education and Research

Foundation, and the Lam Chung Nin Foundation for Systems Biomedicine at

Peking University.

Received: June 17, 2015

Revised: September 8, 2015

Accepted: October 5, 2015

Published: November 5, 2015

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