| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 32, 30051-30056, August 8, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||
From the
Graduate Program in Tumor Biology, Mayo
Graduate School, Rochester, Minnesota 55905, the
Divisions of Oncology Research and Molecular
Pharmacology and Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, and
the ¶School of Medicine, University of Puerto
Rico, San Juan, Puerto Rico 00936
Received for publication, November 13, 2002 , and in revised form, May 21, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although the role of cohesion in the metaphase-anaphase transition is
relatively well understood, there is comparatively little information about
the establishment of cohesion. It is known that this process occurs during S
phase and presumably is linked to the replication fork
(1–4).
In addition, it is thought to involve a polymerase switch analogous to the one
mediated by the replication factor C
(RFC)1 complex during
lagging strand DNA synthesis
(10). RFC functions by loading
the processivity factor PCNA onto DNA and mediating the switch from DNA
polymerase 
to DNA polymerase 
. A novel DNA polymerase, DNA
polymerase 
(8),
initially named DNA polymerase 
(9), has recently been
described. This novel polymerase is thought to mediate DNA synthesis over
areas bound to cohesion proteins, but the mechanism of recruitment of this
polymerase is unknown. Presumably, DNA polymerase relocates to
replication forks with the aid of RFC or a related clamp loader complex. This
alternative clamp loader may be a recently described complex consisting of
RFC2–5 and CTF18/Chl12, identified in Saccharomyces cerevisiae
(5–7).
Two additional proteins, CTF8 and DCC1, were also shown to interact with CTF18
in this complex (5). It has
been hypothesized that CTF18, CTF8, and DCC1 form part of a clamp loader
complex that tethers an unidentified clamp to precohesion areas of DNA,
resulting in the recruitment of DNA polymerase . The passage of
polymerase over the precohesion area coincides with cohesion
establishment, suggesting that the replication fork plays an active role in
this process (10). There is
evidence that polymerase recruitment depends on the
CTF18/CTF8/DCC1-containing complex. In yeast, mutation of any of these three
proteins resulted in precocious sister chromatid separation, increased rates
of chromosome loss, and synthetic lethality with mutations in cohesion
proteins such as SCC1 and SMC3
(5–7).
Here, we describe the cloning and the initial characterization of this
evolutionary conserved alternative RFC complex in Homo sapiens. The
three proteins, which we designate hCTF18, hCTF8, and hDCC1, show the same
pattern of association in humans as in S. cerevisiae. This suggests a
high degree of conservation between yeast and humans in the machinery of
cohesion establishment.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Antibodies—We used two complementary methods for antibody
production. Most of our antibodies were obtained from synthetic peptides
selected for maximum antigenicity. These peptide sequences were
CKRTRDEVDATLQIAKLNAAE (hDCC1-N), CQNGVKVYNSRRPIS (hDCC1-C),
CKDKILFKTRPKPIITSVPKKV (hCTF8-C), EEMEEPPPPDSSPTDITPPPSPEDC (hCTF18-A),
HERPSRKDRPSVEPARVSKEC (hCTF18-C), CAPRNHEQRLEHIMRRAAREEQPEK (hCTF18-D),
CNIQQKTDEKVDESGPPAPSK and CPRRGRRPKSESQGNATKND (hPds5); CKRKRGRPGRPPSTNKKPRKS
and CSSSSKTSSVRNKKGRPPLHKKR (SA1); and CSSRGSTVRSKKSKPSTGKRKVV and
CDLPPSKNRRERTELKPDFFD (SA2). Solutions of these peptides coupled to
mariculture keyhole limpet hemocyanin (Pierce) were used for rabbit polyclonal
antibody production. Antibodies recognizing hDCC1 (hDCC1–4,
hDCC1–5, and hDCC1–6) were produced from different
glutathione S-transferase-fused DCC1 protein fragments. hDCC1 cDNA
was amplified via reverse transcription-PCR (Titan System; Roche Applied
Science) from RNA isolated from HBL-100 cells. The sequences were subcloned
into a pGEX-4T-1 vector, which was then transformed into competent BL21 codon
+ cells. Following induction of BL21 codon + cells with 0.4 mM
isopropyl-
-D-thiogalactopyranoside, glutathione
S-transferase fusion proteins were bound to glutathione-conjugated
Sepharose beads and eluted with 5 mM glutathione. Eluates were used
for rabbit polyclonal antibody production following standard procedure. All of
the antibodies were affinity-purified with protein/peptide-conjugated
Sepharose bead columns.
Immunoprecipitation—The cells were harvested, washed with PBS, and lysed with 1x NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.5% Nonidet P-40) at 4 °C for 10 min. Some experiments were repeated using a gentler lysis buffer (11) containing 10 mM HEPES, pH 7.4, 150 mM KCl, 10 mM MgCl2, and 0.1% Triton X-100. For immunoprecipitation, 200 µl of whole cell lysate was incubated with 2 µg of antibody and 20 µl of protein A-Sepharose beads (Sigma) on a shaker at 4 °C for 90 min. Following three washes with lysis buffer, the immunoprecipitates were fractionated via SDS-PAGE (10% gel). The proteins were then transferred to an Immobilon-PTM polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were incubated with 1:1000 dilutions of primary antibody and 1:1000 dilutions of protein A-horseradish peroxidase and were developed using SuperSignal chemoluminescent solution (Pierce). Selected membranes were stripped with 7 M guanidine hydrochloride for 30 min and reblotted.
Cell Culture and Synchronization—MCF-7, T24, and HeLa cells were cultured in 10-cm plates with either Dulbecco's modified Eagle's medium or RPMI 1640 medium containing 10% fetal calf serum and 1% 10,000 units/ml of penicillin/streptomycin. T24 cells were synchronized by contact inhibition. They were released into the cell cycle after 4 days by subculturing in a 1:4 ratio and harvested at selected time points. HeLa cells were synchronized by drug treatment. S phase fractions were obtained either by a 24-h treatment with 1 mM hydroxyurea (Sigma) or a double block with 2 mM thymidine (Sigma). The cells were treated with thymidine for 17 h, given fresh medium for 10 h, retreated with thymidine for 19 h, and then harvested. M phase fractions were obtained either by treatment with 10 ng/ml nocodazole (Sigma) or by agitating plates of normally cycling cells and harvesting the nonadherent mitotic fraction. In all cases, cell cycle distribution was verified by fluorescence automated cell sorting.
Immunofluorescence—HeLa cells were grown to 20% confluence
on glass slides affixed to the bottom of 6-well plates. They were fixed with
3% paraformaldehyde for 20 min at 4 °C, permeabilized with 1% Triton X-100
with 0.5% Nonidet P-40 for 30 min, and blocked with 5% milk in PBS for a
minimum of 3 h at 4 °C. The cells were then incubated in affinity-purified
primary antibody (PC-10 PCNA and either hCTF18-D or
hDCC1-N) and 5% goat serum with PBS for4hat4 °C. Immune complexes
were detected with rhodamine-conjugated goat anti-rabbit (Jackson) and
fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson) in 5% goat
serum with PBS. Chromatin was counterstained with 2 µg/ml Hoechst 33342 in
PBS for 30 s. Stained cells were analyzed with a Nikon ECLIPSE E800
fluorescence microscope.
Cell Fractionation—To verify subcellular CTF18 localization and to determine the affinity of CTF18 for chromatin at different stages of the cell cycle, HeLa cells were fractionated using a protocol modified slightly from Méndez and Stillman (13). HeLa cells were synchronized as described above, harvested, and resuspended in PBS to equalize cell concentration. The cells were first treated with a nonionic detergent (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 1 mM dithiothreitol, 10% glycerol, and 0.1% Triton X-100 supplemented with a protease inhibitor mixture (Roche Applied Science)) for 5 min on ice to isolate nuclei. Nuclei were spun down at 1300 x g for 4 min at 4 °C, and the supernatant (cytoplasmic fraction) was clarified at 20,000 x g for 20 min at 4 °C. Isolated nuclei were either directly lysed with nuclear lysis buffer (3 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol, and protease inhibitors), or pretreated with 1 unit of micrococcal nuclease to release chromatin-bound proteins. Nuclease treatment was performed in nuclear isolation buffer supplemented with 1 mM CaCl2 and micrococcal nuclease (Sigma) for 1 min at 37 °C. The reaction was quenched with 1 mM EGTA. The supernatant from this reaction was then clarified by high speed centrifugation. Untreated nuclei were lysed with nuclear lysis buffer for 30 min on ice and centrifuged for 5 min at 3000 x g. The nuclear lysate was clarified with high speed centrifugation.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
hCTF18, hCTF8, and hDCC1 Form a Complex in the Cell—To
confirm that these putative human homologs of yeast CTF8/CTF18/DCC1 do form a
complex in mammalian cells, we generated multiple rabbit polyclonal antibodies
against hCTF18, hCTF8, and hDCC1 (Fig.
2A). hDCC1 immunoprecipitates contained a band migrating
at 
45 kDa, which is consistent with the predicted molecular mass for
hDCC1 of 44.8 kDa. hCTF18 immunoprecipitates contained a band near 110 kDa,
consistent with the theoretical molecular mass for hCTF18 of 110.2 kDa.
|
To determine whether these three proteins interact in humans, we
immunoprecipitated MCF-7 whole cell lysates with hDCC1-C,
hCTF18-C, and hCTF8-C and immunoblotted with hCTF18-D or
hDCC1-N antibodies. As shown in Fig.
2B, all three proteins associated with each other,
suggesting that a complex containing hCTF18, hCTF8, and hDCC1 does exist in
human cells.
The Expression of hCTF18 and hDCC1 Is Cell Cycle-regulated—Because hCTF18, hCTF8, and hDCC1 may be involved in cohesion establishment during S phase in humans, we examined whether the expression of these proteins might be regulated in a cell cycle-dependent manner. We analyzed hDCC1 and hCTF18 protein levels in T24 cells that were grown to confluence (arrested by contact inhibition at G0 phase). After 4 days, the cells were trypsinized, replated in fresh medium, and harvested at various time points. As shown in Fig. 3, hDCC1 and hCTF18 protein levels are undetectable at 0 and 8 h after replating. In contrast, the proteins are expressed at low levels as cells enter S phase (24 h), and expression increases dramatically as cells progress through S phase. These data suggest that hDCC1 and hCTF18 are expressed only in actively dividing cells, consistent with their potential role in cohesion establishment in S phase.
|
hCTF18, hCTF8, and hDCC1 Associate with RFC3—S. cerevisiae CTF18, CTF8, and DCC1 have been shown to interact with all four small RFC subunits. We next examined whether the human homologs would show similar interactions with RFC subunits. As shown in Fig. 4A, the p38 small subunit of replication factor C co-immunoprecipitated hCTF18, hCTF8, and hDCC1 and vice versa. Thus, it is likely that hCTF18, hCTF8, and hDCC1 form an alternative RFC complex similar to that observed in yeast.
|
The hCTF18/hCTF8/hDCC1-containing Complex
Co-immunoprecipitates with PCNA but Not the Rad9/Rad1/Hus1
Complex—Because the hCTF18/hCTF8/hDCC1-containing RFC complex is
likely to function as a clamp loader complex, we sought to determine whether
it interacts with any known DNA clamps. These include PCNA, which is involved
in DNA synthesis, and the Rad9/Rad1/Hus1 complex, which is involved in the DNA
damage response. We immunoprecipitated hCTF18, PCNA, and p38 RFC (as positive
control) and Western blotted with PCNA antibody. As shown in
Fig. 4B (top
panel), PCNA co-immunoprecipitated with p38 or hCTF18-C. As a negative
control, rabbit IgG did not immunoprecipitate PCNA. Conversely, hCTF18 also
co-immunoprecipitated with PCNA (Fig.
4B, bottom panel). However, only a very small
amount of PCNA co-immunoprecipitated with hCTF18, leading us to speculate that
PCNA may not be the clamp primarily associated with CTF18/CTF8/DCC1.
Therefore, we performed similar experiment to look for an interaction with the
Rad9/Rad1/Hus1 clamp. The Rad9/Rad1/Hus1 complex forms a PCNA-like clamp
structure and is believed to be involved in DNA damage responses. We failed to
detect any stable interaction between hCTF18/hCTF8/hDCC1 and the
Rad9/Rad1/Hus1 complex (Fig.
4C and data not shown). As a positive control, we readily
detected the interaction between Rad17 and the Rad9/Rad1/Hus1 complex
(11). In summary, these
results show that hCTF18/hCTF8/hDCC1 selectively binds PCNA. This finding is
supported by a recent paper that identified the putative hCTF18/RFC2–5
complex as a PCNA-binding partner through a proteomics approach
(14). However, we cannot rule
out that the hCTF18/hCTF8/hDCC1 complex may associate with another yet
unidentified alternative clamp, which may be functionally redundant with PCNA
under certain conditions.
hCTF18, hCTF8, and hDCC1 Do Not Stably Associate with Cohesin Subunits—Because CTF18, CTF8, and DCC1 are thought to be involved in the establishment of sister chromatid cohesion in yeast, we next examined whether their human homologs interact with proteins in the cohesin complex. These include SMC1, SMC3, Pds5, and the two human SCC3 homologs SA1 and SA2. As shown in Fig. 4D, we failed to detect any stable interaction with members of the cohesin complex. Equivalent experiments in S. cerevisiae also failed to find a physical interaction of CTF18/CTF8/DCC1 with cohesion proteins (5). It is possible that this alternative RFC complex does not directly interact with cohesion proteins or that this interaction may be very transient.
hCTF18, hCTF8, and hDCC1 Are Predominantly Nuclear
Proteins—We used immunofluorescence microscopy to determine the
subcellular localization of hCTF18 and hDCC1. The proteins had a very similar
staining pattern. They localized to nuclear heterochromatin in a diffuse
manner and appeared less abundant in nucleoli. Their distribution did not
appear to change over the cell cycle, except during mitosis, when hCTF18 and
hDCC1 redistributed throughout the entire cell. S phase cells, identified by
co-immunostaining with PCNA (not shown), did not appear noticeably
different in terms of hCTF18/hDCC1 staining when compared with non-S phase
cells. hCTF18 Preferentially Associates with Chromatin during S
Phase—Because we hypothesize that hCTF18 and hDCC1 may form part of
an RFC-containing alternative clamp loader complex, we investigated whether
its putative S phase function correlates with chromatin binding. Using a cell
fractionation assay, we compared protein localization in HeLa cells arrested
at different phases of the cell cycle. Whole cell lysate shows that hCTF18
protein level remains stable in actively cycling cells, regardless of the cell
cycle stage. We collected mitotic cells by mitotic shake-off and S phase cells
by synchronization using thymidine block or hydroxyurea treatment (see
"Materials and Methods" for details). In mitotic cells, hCTF18 is
present in the initial lysate, suggesting that the complex does not remain
associated with chromatin during mitosis. However, only small amounts of
hCTF18 are readily extractable from the nucleus during S phase. When S phase
nuclei are treated with micrococcal nuclease to release chromatin-bound
proteins, the majority of hCTF18 becomes solubilized. This result strongly
suggests that hCTF18 associates with chromatin, not with the nuclear matrix,
during S phase. To verify the integrity of the fractionation assay, we also
blotted with hOrc2p, one of the chromatin-associated proteins studied
by the original authors using this fractionation assay
(13). Finally, we reblotted
with anti--actin as a loading control for whole cell and cytoplasmic
fractions and with anti-CREB1 as a loading control for nuclear extracts
(Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Despite many recent advances, the mechanism of sister cohesion
establishment remains largely unknown. As reviewed by Carson and Christman
(10), cohesion establishment
appears to involve a polymerase switch from DNA polymerase to the
newly described DNA polymerase (formerly polymerase ). This
switch is probably analogous to the polymerase /polymerase
switch mediated by RFC-PCNA during Okazaki fragment synthesis
(12). The novel clamp loader
complex hCTF18/hCTF8/hDCC1/RFC
(2–5)
may replace RFC during replication over cohesin-bound DNA and associates with
PCNA. However, it is not clear whether PCNA is the clamp preferentially bound
by this novel complex or whether RFC(hCTF18/hCTF8/hDCC1) can recruit an
additional clamp-like structure. Here, we have shown that there is no
detectable association of hCTF18/hCTF8/hDCC1 with the hRad9/hRad1/hHus1
complex, a PCNA-like complex involved in the DNA damage response
(15). However, there are other
proteins such as CTF7 that genetically interact with CTF18 and may form a
PCNA-like clamp structure. The function of PCNA appears to overlap with that
of CTF7, because overexpression of POL30 (PCNA) in yeast rescues the lethal
phenotype of a temperature-sensitive mutant of CTF7
(16).
Mayer et al. (5) noted that CTF18, CTF8, and DCC1 are excellent candidate genes for cancer predisposition, because their loss leads to an abnormal yet viable phenotype. If hCTF18/hCTF8/hDCC1 proves to be functionally as well as structurally equivalent to its S. cerevisiae homologs, it is likely that alterations of this complex may play a role in human disease. One of the earliest stages of tumor progression is the loss of genomic stability (17). Indeed, 80–90% of solid tumors are highly aneuploid, and this aneuploidy is thought to arise before frank transformation takes place. Therefore, genes that mediate chromosomal stability are potential early mutational targets in cancer. It remains to be determined whether the members of this novel RFC-containing complex are mutated in human malignancies or whether their loss can contribute to genomic instability. Further study of this novel replication factor C-containing complex will provide much needed insight into the poorly understood mechanism of sister chromatid cohesion establishment in humans. In addition, it may lead to a better understanding of the mechanism of aneuploidy during human carcinogenesis.
|
FOOTNOTES |
|---|
|| To whom correspondence should be addressed: Div. of Oncology Research, Guggenheim Bldg., Rm. 1306, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Tel.: 507-538-1545; Fax: 507-284-3906; E-mail: chen.junjie{at}mayo.edu.
1 The abbreviations used are: RFC, replication factor C; CTF, chromosomal
transmission fidelity; DCC, defective in sister chromatid cohesion; PCNA,
proliferating cell nuclear antigen, SMC, structural maintenance of
chromosomes; PBS, phosphate-buffered saline.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  K. M. Berkowitz, K. H. Kaestner, and T. A. Jongens Germline expression of mammalian CTF18, an evolutionarily conserved protein required for germ cell proliferation in the fly and sister chromatid cohesion in yeast Mol. Hum. Reprod., March 1, 2008; 14(3): 143 - 150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. B. Ansbach, C. Noguchi, I. W. Klansek, M. Heidlebaugh, T. M. Nakamura, and E. Noguchi RFCCtf18 and the Swi1-Swi3 Complex Function in Separate and Redundant Pathways Required for the Stabilization of Replication Forks to Facilitate Sister Chromatid Cohesion in Schizosaccharomyces pombe Mol. Biol. Cell, February 1, 2008; 19(2): 595 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. O. Bylund and P. M. J. Burgers Replication Protein A-Directed Unloading of PCNA by the Ctf18 Cohesion Establishment Complex Mol. Cell. Biol., July 1, 2005; 25(13): 5445 - 5455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Game, M. S. Williamson, and C. Baccari X-Ray Survival Characteristics and Genetic Analysis for Nine Saccharomyces Deletion Mutants That Show Altered Radiation Sensitivity Genetics, January 1, 2005; 169(1): 51 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Petronczki, B. Chwalla, M. F. Siomos, S. Yokobayashi, W. Helmhart, A. M. Deutschbauer, R. W. Davis, Y. Watanabe, and K. Nasmyth Sister-chromatid cohesion mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the helicase Chl1 and the polymerase-{alpha}-associated protein Ctf4 is essential for chromatid disjunction during meiosis II J. Cell Sci., July 15, 2004; 117(16): 3547 - 3559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Shiomi, A. Shinozaki, K. Sugimoto, J. Usukura, C. Obuse, and T. Tsurimoto The reconstituted human Chl12-RFC complex functions as a second PCNA loader Genes Cells, April 1, 2004; 9(4): 279 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Warren, D. M. Eckley, M. S. Lee, J. S. Hanna, A. Hughes, B. Peyser, C. Jie, R. Irizarry, and F. A. Spencer S-Phase Checkpoint Genes Safeguard High-Fidelity Sister Chromatid Cohesion Mol. Biol. Cell, April 1, 2004; 15(4): 1724 - 1735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Mayer, I. Pot, M. Chang, H. Xu, V. Aneliunas, T. Kwok, R. Newitt, R. Aebersold, C. Boone, G. W. Brown, et al. Identification of Protein Complexes Required for Efficient Sister Chromatid Cohesion Mol. Biol. Cell, April 1, 2004; 15(4): 1736 - 1745. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
