Mediator of DNA Damage
Checkpoint Protein 1 Regulates
BRCA1 Localization and
Phosphorylation in DNA Damage
Received for publication, February 10, 2003,
and in revised form, February 25, 2003
Published, JBC Papers in Press, February 27, 2003,
Zhenkun Lou‡, Claudia Christiano Silva Chini‡,
Katherine Minter-Dykhouse, and Junjie Chen§
From the Department of Oncology, Mayo Clinic and
Foundation, Rochester, Minnesota 55905
BRCA1 is a tumor suppressor involved in DNA repair
and damage-induced checkpoint controls. In response
to DNA damage, BRCA1 relocalizes to nuclear foci at the
sites of DNA lesions. However, little is known about the
regulation of BRCA1 relocalization following DNA dam-
age. Here we show that mediator of DNA damage check-
point protein 1 (MDC1), previously named NFBD1 or
Kiaa0170, is a proximate mediator of DNA damage re-
sponses that regulates BRCA1 function. MDC1 regulates
ataxia-telangiectasia-mutated (ATM)-dependent phos-
phorylation events at the site of DNA damage. Impor-
tantly down-regulation of MDC1 abolishes the relocal-
ization and hyperphosphorylation of BRCA1 following
DNA damage, which coincides with defective G2/M
checkpoint control in response to DNA damage. Taken
together these data suggest that MDC1 regulates BRCA1
function in DNA damage checkpoint control.
FHA1and BRCT domains are functional modules that are
involved in protein-protein interaction (1–3). Many proteins
involved in the DNA damage response pathway, such as mam-
malian BRCA1, 53BP1, Chk2, NBS1, yeast Rad9, and Rad53,
contain FHA or BRCT domains. Furthermore mutations within
FHA and BRCT domains have been associated with tumorigen-
esis (4).2These findings suggest important roles of FHA and
BRCT domains in DNA damage response pathways.
Kiaa0170 or NFBD1 (nuclear factor with BRCT domains
protein 1) is a nuclear protein that contains both FHA and
BRCT domains. Our initial studies on Kiaa0170 and studies
from other laboratories have shown that Kiaa0170 forms nu-
clear foci at the sites of DNA damage and is phosphorylated in
an ATM-dependent manner (5–7). Furthermore Kiaa0170
functions as a critical regulator in DNA damage signaling
pathways (7, 8).3Therefore, Kiaa0170 has been renamed as
mediator of DNA damage checkpoint protein 1 (MDC1) to bet-
ter reflect its role in DNA damage checkpoint controls.4In this
study, we further explored the role of MDC1 in ATM-dependent
DNA damage response pathways.
Plasmids and Small Interfering RNAs (siRNAs)—MDC1 cDNA was
kindly provided by Dr. T. Nagase from Kazusa DNA Research Institute
(Chiba, Japan). MDC1 siRNAs were synthesized by Xerogon Inc.
(Huntsville, AL). The siRNA duplexes were 21 base pairs including a
two-deoxynucleotide overhang. The coding strand of MDC1 siRNA1 was
UCCAGUGAAUCCUUGAGGUdTdT, and the coding strand of MDC1
siRNA2 was ACAACAUGCAGAGAUUGAAdTdT. The coding strand of
BRCA1 siRNA was GGAACCUGUCTCCACAAAGdTdT, and the con-
trol siRNA was UUCAAUAAAUUCUUGAGGUdTdT.
Antibodies and Cell Lines—MDC1 antibodies were raised against
glutathione S-transferase fusion proteins containing N-terminal resi-
dues 1–150 or 151–484 of MDC1. Anti-phospho-H2AX (?H2AX) and
anti-BRCA1 antibodies were generated as described previously (8).
Anti-p1524BRCA1 antibodies were kindly provided by Dr. KumKum
Khanna. Anti-pS317Chk1 antibodies and anti-pATM/ATR were pur-
chased from Cell Signaling. Anti-Chk1 mAb was purchased from Santa
Cruz Biotechnology. Anti-phospho-histone 3 antibodies were purchased
from Upstate Biotechnology. All cells were obtained from American
Type Culture Collection and cultured in RPMI 1640 medium supple-
mented with 10% fetal bovine serum.
Immunoprecipitation, Immunoblotting, and Immunostaining—Cell
lysate preparation, immunoprecipitation, immunoblotting, and immu-
nostaining were performed as described before (8).
siRNA Transfection—siRNA transfection was performed as de-
scribed previously (9). Briefly, cells were grown in six-well plate to 30%
confluence and immediately before transfection washed with serum-
free medium, and 800 ?l of serum-free medium were added per well. For
each well, 200 nM siRNA was mixed with 5 ?l of Oligofectamine (In-
vitrogen) in 200 ?l of serum-free medium. The mixtures were incubated
for 20 min at room temperature and then added to cells. Serum was
added 4 h later to a final concentration of 10%. 24 h after the initial
transfection, a second transfection was performed in the same way as
the previous one. 72 h after initial transfection, cells were treated and
harvested as indicated.
Phospho-H3 Staining—siRNA-transfected cells were ? irradiated (2
Gy) or left untreated. Cells were then stained with anti-phospho-H3
antibodies, and phospho-H3-positive cells were evaluated by counting
or fluorescence-activated cell sorter.
RESULTS AND DISCUSSION
To test the role of MDC1 in ATM-dependent phosphorylation
events, we used siRNA technology (9) to down-regulate MDC1
(Fig. 1A) and monitored ATM-dependent phosphorylation
events by immunofluorescence staining (Fig. 1B). An antibody
raised against phosphoepitope substrates of ATM or ATR has
been shown to specifically recognize ATM/ATR-dependent
phosphorylation events (10). As shown in Fig. 1B, in cells
transfected with control siRNA, ionizing radiation (IR)-induced
nuclear foci were present in most irradiated cells, suggesting
the accumulation of phosphorylated ATM substrates at the
* This work was supported in part by National Institutes of Health
Grants NIH RO1 CA89239 and CA92312 and by the Prospect Creek
Foundation and the Breast Cancer Research Foundation. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Both authors contributed equally to this work.
§ Recipient of Department of Defense Breast Cancer Career Devel-
opment Award DAMD17-02-1-0472. To whom correspondence should be
addressed: Dept. of Oncology, Mayo Clinic, Guggenheim Bldg., Rm.
1342, 200 First St. S. W., Rochester, MN 55905. Tel.: 507-538-1545;
Fax: 507-284-3906; E-mail: email@example.com.
1The abbreviations used are: FHA, forkhead-associated; BRCT,
BRCA1 C-terminal; MDC1, mediator of DNA damage checkpoint pro-
tein 1; ATM, ataxia-telangiectasia-mutated; ATR, ATM and Rad3-re-
lated; siRNA, small interfering RNA; H3, histone 3; Gy, gray; IR,
ionizing radiation; ?H2AX, phospho-H2AX; BARD1, BRCA1-associated
2Breast Cancer Information Core at research.nhgri.nih.gov/bic/ on
the World Wide Web.
3S. Jackson and S. Elledge, personal communication.
4S. J. Elledge and S. P. Jackson, personal communication.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 278, No. 16, Issue of April 18, pp. 13599–13602, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
by guest on November 6, 2015