JBC Papers in Press. Published on November 21, 2016 as Manuscript M116.

760561
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.760561

The MMR system and CAF-1

The major replicative histone chaperone CAF-1 suppresses the activity of the DNA mismatch repair
system in the cytotoxic response to a DNA methylating agent
Lyudmila Y. Kadyrova, Basanta K. Dahal, and Farid A. Kadyrov1
From the Department of Biochemistry and Molecular Biology, Southern Illinois University School of
Medicine, Carbondale, IL 62901, USA
Running title: The MMR system and CAF-1
1

To whom correspondence should be addressed: Dr. Farid A. Kadyrov, Department of Biochemistry

and Molecular Biology, Southern Illinois University School of Medicine, Neckers Building, 1245
Lincoln Drive. Carbondale, IL 62901, USA, Telephone: (618) 453-6405; FAX: (618) 453-6440; Email: fkadyrov@siumed.edu

1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

Keywords: DNA mismatch repair, genomic instability, cancer, mutL homolog 1 (MLH1), DNA
replication, histone chaperone, CAF-1.
__________________________________________________________________________________
against killing by the Sn1-type methylating
ABSTRACT
The DNA mismatch repair (MMR)
agent.
system corrects DNA mismatches in the
The DNA mismatch repair (MMR)
genome. It is also required for the cytotoxic
system
has been conserved from bacteria to
response
of
O6-methylguanine-DNA
humans as a consequence of its importance for
methyltransferase
(MGMT)-deficient
the maintenance of genome stability (1-3). The
mammalian cells and yeast mgt1 rad52 cells
MMR system has several activities that are
to treatment with Sn1-type methylating agents,
involved in genome metabolism (1,3-10). The
which produce cytotoxic O6-methylguanine (O6correction of DNA mismatches is the best
mG) DNA lesions. Specifically, processing of
understood activity of the MMR system.
irreparable O6-mG-containing mispairs by the
Eukaryotic MMR leads to the correction of
MMR system causes DNA degradation,
errors caused by the replication fork DNA
triggering cell death; this process forms the
polymerases , , and , and it also changes the
basis of treatments of MGMT-deficient
outcome of homologous recombination (11-17).
cancers with Sn1-type methylating drugs.
A considerable number of proteins have been
Recent research supports the view that
suggested to contribute to or regulate eukaryotic
processing of irreparable O6-mG-containing
MMR (6,18-20). Strong evidence indicates the
mispairs by the MMR system and CAF-1primary mismatch recognition factor MutS
dependent packaging of the newly replicated
(MSH2-MSH6 heterodimer), MutL (MLH1DNA into nucleosomes are two concomitant
PMS2 heterodimer in humans and Mlh1-Pms1
processes that interact with each other. Here,
heterodimer in budding yeast) endonuclease, the
we studied whether CAF-1 modulates the
replicative clamp PCNA (proliferating cell
activity of the MMR system in the cytotoxic
nuclear antigen), the clamp loader RFC
response to Sn1-type methylating agents. We
(replication factor C), the exonuclease EXO1,
found that CAF-1 suppresses the activity of the
the secondary mismatch recognition factor
MMR system in the cytotoxic response of yeast
MutS (MSH2-MSH3 heterodimer), and the
mgt1 rad52 cells to the prototypic Sn1-type
replicative DNA polymerase (Pol ) play
methylating
agent
N-methyl-N'-nitro-Nmajor roles in eukaryotic MMR (21-39).
nitrosoguanidine (MNNG). We also report
Several eukaryotic MMR reactions have
evidence that in human MGMT-deficient cellbeen described (10,36,40-43). One of these
free extracts CAF-1-dependent packaging of
reactions removes mismatches on both the 3irreparable O6-mG-T mispair-containing DNA
and 5-nicked DNA molecules in an excisioninto nucleosomes suppresses its degradation by
dependent manner and is probably necessary for
the MMR system. Taken together, these findings
the majority of MMR events in wild-type cells
suggest that CAF-1-dependent incorporation of
(36). This MMR reaction depends on the
irreparable O6-mG-T mispair-containing DNA
activities of MutS, MutL, PCNA, RFC,
into nucleosomes suppresses its degradation by
EXO1, RPA, and Pol (32,33,35-37,41) and is
the MMR system, thereby defending the cell
initiated by recognition of the mismatch by

The MMR system and CAF-1

RESULTS
CAF-1 suppressed the activity of the MMR
system in the cytotoxic response of yeast mgt1
rad52 cells to MNNG
The cytotoxic response to Sn1-type
methylating agents occurs in the chromatin
environment (46,52,53,68). However, it has
remained unknown whether the chromatin
environment affects the cytotoxic response to
Sn1-type methylating agents. It has also been
unknown whether histone chaperones, proteins
that are involved in the control of chromatin
environment, influence the cytotoxic response to
Sn1-type methylating agents. We wanted to
study whether the major replicative histone
chaperone CAF-1 impacts the cytotoxic response

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MGMT due to methylation of the MGMT

promoter (50). MGMT-deficient cancer cells
treated with the Sn1-type methylating agent
accumulate O6-mG-T mispairs that are
recognized by MutS (51). Upon recognition of
an O6-mG-T mispair, MutS initiates its repair.
If the O6-mG is on the discontinuous strand, it
gets repaired (52). In contrast, if the O6-mG is on
the continuous strand, it triggers futile cycles of
MMR (52). The futile cycles of MMR lead to
the formation of persistent strand breaks (53)
which are converted in the next S phase into
double-strand breaks that cause cell cycle arrest
followed by cell death (54,55). Consistent with
this, double-strand break repair defects sensitize
eukaryotic cells to the killing effects of Sn1-type
methylating agents (47,56).
The heterotrimeric CAF-1 is the major
histone chaperone for the assembly of
nucleosomes onto the newly replicated DNA
(57-61). CAF-1 loads histone (H3-H4)2
tetramers onto DNA, producing tetrasomes
(62,63). Each tetrasome is then converted into a
nucleosome by the addition of two histone H2AH2B dimers (57,64). CAF-1 interacts physically
with PCNA and this interaction is necessary for
the action of CAF-1 on the newly replicated
DNA (65,66). Recent research has indicated that
postreplicative MMR coincides with CAF-1dependent nucleosome assembly and that the
two processes interact with each other
(43,63,67). Furthermore, recent findings are
consistent with the idea that the eukaryotic
MMR system degrades irreparable O6-mG-T
mispair-containing DNA when it is packaged
into nucleosomes by the CAF-1-dependent
mechanism (53,63,67). We show here that CAF1 suppresses the activity of the MMR system in
the cytotoxic response to Sn1-type methylating
agents.

MutS (21,22). After recognizing the mismatch,

MutS cooperates with RFC-loaded PCNA to
activate MutL endonuclease (37,41,44). The
activated MutL endonuclease incises the
discontinuous daughter strand. An incision that
is generated by MutL endonuclease 5 to the
mismatch is used by EXO1 to enter the DNA
and excise the mismatch in a 53-directed
reaction modulated by MutS and RPA
(33,35,37,45). A gap generated by EXO1 action
is filled in by Pol holoenzyme. Though Pol
holoenzyme can also perform gap filling in a
reconstituted excision-dependent MMR reaction,
the specific activity of this enzyme is much
lower than that of Pol holoenzyme (43). The
mutator phenotype of an exo1 mutant is weaker
compared to the mutator phenotype of an msh2
or mlh1 mutant, indicating that the MMR
system can remove DNA polymerase errors in
EXO1-deficient cells (14,28,34). In agreement
with this idea, an excision-independent MMR
reaction that involves MutS, MutL, PCNA,
RFC, RPA, and Pol has been described (41).
Like the excision-dependent MMR reaction, the
excision-independent MMR reaction requires the
MutL endonuclease activity for incision of the
discontinuous daughter strand. After MutL
endonuclease incises the discontinuous daughter
strand, the Pol holoenzyme uses a MutLgenerated 3 end that is 5 to the mismatch to
perform strand-displacement DNA synthesis that
removes the mismatch. The role of the Pol
holoenzyme in the excision-independent MMR
reaction may be unique because the replacement
of the Pol holoenzyme with the Pol
holoenzyme abolishes the excision-independent
MMR reaction (43).
One of the activities of the MMR system
is required for the cytotoxic response of MGMT
(O6-methyl guanine methyl transferase)-deficient
mammalian cells and yeast mgt1 rad52 cells
to Sn1-type methylating agents (46,47). This
activity of the MMR system depends on MutS
and MutL and is necessary for several
therapies against MGMT-deficient cancers (3).
The
treatment
involves
dacarbazine,
procarbazine, or temozolomide, each of which is
an Sn1-type methylating drug that triggers death
of MGMT-deficient cancer cells by activating
the MMR system. O6-methylguanine (O6-mG) is
the cytotoxic product of treatment of the cell
with the Sn1-type methylating agent (48).
Normally, O6-mG lesions are removed by
MGMT that protects the cell from killing by the
Sn1-type methylating agent (49). However, a
significant number of cancers are deficient in

The MMR system and CAF-1

independent mechanisms. Nevertheless, there is
a significant similarity between one of these
drugs, camptothecin, and MNNG in that doublestrand breaks caused by these two agents are
formed during DNA replication. The results of
our genetic experiments (Figure 1A and 1C)
raised a possibility that loss of CAC1 sensitized
the yeast mgt1 rad52 cells to the cytotoxic
effects of DNA damaging agents that generate
DNA breaks. To address this possibility, we
studied the effect of CAC1 absence on the
sensitivity of the mgt1 rad52 cells to
camptothecin, hydroxyurea, and bleomycin
(Figure 1D and 1E). An analysis of the data
showed that the cac1 mgt1 rad52 cells and
the mgt1 rad52 cells had the same
sensitivities to bleomycin, camptothecin, and
hydroxyurea. Thus, CAC1 absence did not affect
the sensitivity of the yeast mgt1 rad52 cells to
bleomycin, camptothecin, and hydroxyurea,
drugs that kill cells via MMR systemindependent mechanisms. Because MNNG kills
the yeast mgt1 rad52 cells by activating an
MMR system-dependent mechanism and
bleomycin, camptothecin, and hydroxyurea kill
the cells via other mechanisms, the results of our
genetic experiments (Figure 1A and 1C-E)
indicated that Cac1 increases the survival of the
MNNG-treated mgt1 rad52 cells by being
involved in a process that suppresses the
cytotoxic activity of the MMR system.
CAC2 codes for the second subunit of
yeast CAF-1 (60). We studied whether deletion
of CAC2 sensitized the mgt1 rad52 cells to
MNNG (Figure 1B). The data showed that the
cac2 mgt1 rad52 cells were more sensitive
to MNNG than the mgt1 rad52 cells (Figure
1B-C). A comparison of the sensitivities of the
cac2 msh2 mgt1 rad52, msh2 mgt1
rad52, cac2 mgt1 rad52, and mgt1
rad52 cells (Figure 1C) revealed that MNNG
killed the cac2 mgt1 rad52 cells via an
MMR system-dependent mechanism. As
expected, the cac2 mgt1 rad52 cells were as
sensitive to camptothecin and hydroxyurea as
the mgt1 rad52 cells (Figure 1F). Thus, the
results of these and previous experiments
(Figure 1A-F) demonstrated that CAF-1
increases the survival of MNNG-treated mgt1
rad52 cells by suppressing the cytotoxic
activity of the MMR system.
Loss of HIR2, RTT106, or HHT2-HHF2
did not change the sensitivity of yeast mgt1
rad52 cells to MNNG
Histone H3-H4 chaperone HIR (Hir1Hir2-Hir3-Hpc2 complex) plays a major role in

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to an Sn1-type methylating agent. Previous

research
showed
that
MGMT-deficient
mammalian cells and S. cerevisiae mgt1
rad52 that lack Mgt (the yeast ortholog of
MGMT (69)) and the recombination mediator
Rad52 (70) are efficiently killed by the
prototypic Sn1-type methylating agent MNNG in
a manner that involves the MMR system
(46,47,53). Thus, we utilized the yeast mgt1
rad52 cells to determine whether CAF-1
impacts the cytotoxic response to MNNG. In
budding yeast, CAC1 encodes the largest subunit
of CAF-1 (60). Accordingly, we investigated
whether loss of the CAC1 gene increased the
sensitivity of the yeast mgt1 rad52 cells to
killing by MNNG. The use of an MNNG
cytotoxicity assay permitted us to establish that
the cac1 mgt1 rad52 cells were more
sensitive to treatment with MNNG than the
mgt1 rad52 cells (Figure 1A). A more
detailed analysis revealed that ~1.7% of the
mgt1 rad52 cells and only ~0.2% of the
cac1 mgt1 rad52 cells survived the
treatment with MNNG (Figure 1C). Thus, the
surviving fraction of the MNNG-treated cac1
mgt1 rad52 cells was ~9 times smaller than
that of the MNNG-treated mgt1 rad52 cells.
We then conducted experiments to determine
whether MNNG killed the cac1 mgt1 rad52
cells via an MMR system-dependent mechanism
(Figure 1C).
The results showed that the
deletion of the MMR system gene MLH1
rescued the sensitivity of the cac1 mgt1
rad52 cells to the cytotoxic effect of MNNG.
The experiments also demonstrated that the
mlh1 cac1 mgt1 rad52 cells were as
resistant to the MNNG treatment as the mlh1
mgt1 rad52 cells. Based on these findings,
we concluded that loss of CAC1 sensitizes the
yeast mgt1 rad52 cells to MMR systemdependent killing by MNNG.
The cytotoxicity of Sn1-type methylators
is mediated by replication- and MMR systemdependent double-strand breaks that these agents
form. A number of other DNA damaging agents
generate strand breaks that kill cells. Among
them are camptothecin, hydroxyurea, and
bleomycin. Camptothecin induces replicationdependent double-strand breaks by stabilizing
Topoisomerase I-DNA covalent complexes (71),
hydroxyurea causes double-strand breaks by
depleting the dNTP pools (72), and bleomycin
creates double-strand and single-strand breaks
by attacking DNA (73). Unlike the Sn1-type
methylating agent, camptothecin, hydroxyurea,
and bleomycin kill cells via MMR system-

The MMR system and CAF-1

(Figure 2A) (58). Although the 293T cytosolic
extract lacks the MMR system due to the
absence of MutL (77), supplementation of the
extract with purified MutL reconstitutes the
MMR system (78). In agreement with previous
studies
(37,52,63,77-79),
our
control
experiments showed that (1) the reconstituted
MMR system failed to repair an O6-mG-T
mispair on a nicked DNA (3-nicked O6-mG-T
DNA) but repaired a G-T mispair on a similar
nicked DNA (the 3-nicked G-T DNA) (Figure
3), (2) supplementation of the 293T cytosolic
extract
with
purified
MutL-E705K
endonuclease mutant (37) did not lead to
reconstitution of the MMR system (Figure 3),
(3) the omission of exogenous dNTPs and the
addition of the DNA polymerase inhibitor
aphidicolin led to a near complete inhibition of
the mismatch correction activity of the
reconstituted MMR system (Figure 3), (4) the
293T cytosolic extract had a weak nucleosome
assembly activity (Figure 2B) due to the reduced
level of CAF-1 (Figure 2A).
We next utilized Southern hybridization
to detect MMR system-dependent degradation of
the irreparable O6-mG-T mispair-containing
DNA that was reconstituted with the 293T
cytosolic extract in the absence or presence of
the purified CAF-1 (Figures 4-7). The initial
experiments in this series analyzed degradation
products that were separated on denaturing
agarose gels (Figures 4-6). The data showed that
the reconstituted MMR system degraded the
discontinuous strand of an irreparable O6-mG-T
mispair-containing DNA (the 3-nicked O6-mGT DNA) leading to the formation of an
approximately 130-nt product (Figures 4A and
5A, lane 10). Importantly, the ~130-nt product
was not observed in the reaction mixture that
contained endonuclease-deficient MutL-E705K
instead of MutL (Figures 4A and 5A, lane 13)
demonstrating that the endonuclease activity of
MutL is required for the degradation of the 3nicked O6-mG-T DNA. Additional experiments
revealed that the ~130-nt or a similar product
was not formed from the 3-nicked A-T DNA in
the reaction mixture that contained the
reconstituted MMR system (Figures 4A and 5A,
lane 17). The results of a time course analysis
demonstrated that the ~130-nt product of
degradation of the discontinuous strand of the 3nicked O6-mG-T DNA was observed in the
reaction mixture that was incubated for 10-120
min (Figure 5B). This observation indicated that
the reconstituted MMR system caused persistent
degradation of the discontinuous strand of the 3nicked O6-mG-T DNA. In contrast, the products

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replication-independent nucleosome assembly

(74) and histone H3-H4 chaperone Rtt106
participates in replication-coupled nucleosome
assembly (75). We analyzed whether absence of
HIR2 or RTT106 had an effect on the sensitivity
of the mgt1 rad52 cells to MNNG. The
experiments showed that lack of HIR2 or
RTT106 did not change the sensitivity of the
mgt1 rad52 cells to MNNG (Figure 1G).
The molecular activity of CAF-1 is to
load histone H3-H4 tetramers onto newly
replicated DNA (59,62,64). We wanted to
explore if decreasing histone H3-H4 gene
dosage affected the survival of MNNG-treated
mgt1 rad52 cells. The yeast genome contains
two histone H3-H4 gene loci, HHT1-HHF1 and
HHT2-HHF2. Compared to HHT1-HHF1,
HHT2-HHF2 produces about 5-7 times more
mRNA (76). Either locus is sufficient to
maintain the existence of the yeast cell. We
measured the sensitivity of hht2-hhf2 mgt1
rad52 cells to MNNG and found it to be no
different from that of the mgt1 rad52 cells
(Figure 1G).
CAF-1-dependent
packaging
of
6
irreparable O -mG-T mispair containing DNA
into nucleosomes suppressed its degradation by
the MMR system
MMR system-dependent degradation of
irreparable O6-mG-T mispair-containing DNA is
involved in the cytotoxic response to the Sn1type methylating drug (52,53,68). Our genetic
experiments indicated that CAF-1 activity
suppresses degradation of irreparable O6-mG-T
mispair-containing DNA by the MMR system
(Figure 1). To find evidence that CAF-1dependent incorporation of irreparable O6-mG-T
mispair-containing DNA into nucleosomes
suppresses its degradation by the MMR system,
we performed biochemical experiments that are
summarized in Figures 2-7. DNA substrates that
we utilized in these experiments were 3-nicked
O6-mG-T (3 O6-mG-T), 3-nicked G-T (3 G-T)
and 3-nicked A-T (3 A-T) DNAs.
The
substrates were made using a plasmid, pAH1A,
as a starting material and differed from each
other by 1-2 bases (52). The 3-nicked O6-mG-T
DNA contained a single O6-mG-T mispair, the
3-nicked G-T DNA carried a single G-T
mispair, and the 3-nicked A-T DNA lacked a
mispair. The 3-nicked O6-mG-T DNA was
irreparable by the MMR system because the O6mG was on the continuous strand. In these
biochemical experiments, we used a cytosolic
extract prepared from human embryonic kidney
cell line 293T that lacked MutL and MGMT
(77,78) and had a reduced level of CAF-1

The MMR system and CAF-1

product
(Figure
6A-B).
The
simplest
interpretation of these results is that CAF-1dependent packaging of irreparable O6-mG-T
mispair-containing DNA into nucleosomes
reduces its degradation by the MMR system.
We also utilized Southern hybridization
to analyze the degradation products that were
separated on native agarose gels (Figure 7). The
data revealed that in the 293T extract- and
MutL-containing
reaction
mixture,
a
significant fraction of the 3-nicked O6-mG-T
DNA was converted into a gapped product
(Figure 7). A gap was formed in the
discontinuous but not continuous strand of the
3-nicked O6-mG-T DNA (Figure 7B-C, lane 7).
The same or a similar gapped product was
largely absent in the 293T extract- and MutLE705K-containing mixture (Figure 7B, lane 10).
Although some of the 3-nicked G-T DNA
products in the 293T extract- and MutLcontaining reaction mixture were also gapped
(Figure 7B, lane 2), their yield was ~2.5 times
lower than that of the gapped product of the 3nicked O6-mG-T DNA (Figure 7D). As
expected, 3-nicked A-T DNA products that
were formed in the 293T extract- and MutLcontaining reaction mixture lacked gaps (Figure
7B, lane 12, and Figure 7C). Supplementation of
the 293T extract- and MutL-containing
reaction mixture with 0.6 or 2.4 pmol of purified
CAF-1 caused a significant reduction in the yield
of the gapped product of the 3-nicked O6-mG-T
DNA (Figure 7B-E). This finding supports the
view that CAF-1-dependent packaging of
irreparable O6-mG-T mispair-containing DNA
into nucleosomes suppresses its degradation by
the MMR system.
Degradation of an irreparable O6-mG-T
mispair-containing DNA by the activated MutL
endonuclease in a defined system
Our biochemical experiments (Figures
4-7) have implicated MutL endonuclease
activity in MMR system-dependent degradation
of the discontinuous strand of irreparable O6mG-T mispair-containing DNA. However, these
experiments did not provide a clear view of how
MutL endonuclease activity is involved in this
process. To better understand the involvement of
MutL endonuclease activity in MMR systemdependent degradation of the discontinuous
strand of irreparable O6-mG-T mispaircontaining DNA, we carried out additional
experiments summarized in Figure 8. In these
experiments we studied degradation of the
discontinuous strand of the 3-nicked O6-mG-T
DNA in a defined system (37). Purified proteins
that were included in the defined system were

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of degradation of the discontinuous strand of the

3-nicked G-T DNA that were present in the
reaction mixture incubated for 10 min (Figure
4A, lane 3) were not detected in the same
reaction mixture that was incubated for 60 min
(Figure 5A, lane 3). This finding is consistent
with the view that the product of degradation of
the discontinuous strand of the 3-nicked G-T
DNA is the intermediate of the MMR reaction
(37).
The ~130-nt product was identified by
Southern hybridization with a 32P-labeled probe
that was complementary to a discontinuous
strand sequence located 2-nt downstream from
the mismatched T (Figures 4A and 5A).
However, Southern hybridization with a 32Plabeled probe that was complementary to a
discontinuous strand sequence located 2-nt
upstream from the mismatched T did not detect
the ~130-nt product (Figure 5C). This finding
indicated that the 5 end of the ~130-nt product
was located at or near the mismatch. To
determine whether a different part of the 3nicked O6-mG-T DNA contained a MutL
endonuclease-dependent strand break, we carried
out Southern hybridizations with two other 32Plabeled probes (Figure 5D-E). One of the probes
was complementary to a discontinuous strand
sequence that was downstream from the preexisting strand break (Figure 5D), and the other
to a continuous strand sequence (Figure 5E).
These two Southern hybridizations did not
identify any additional MutL endonucleasedependent strand break in the 3-nicked O6-mGT DNA.
The addition of purified CAF-1 to the
293T extract- and MutL-containing reaction
mixture led to packaging of the 3-nicked O6mG-T DNA into nucleosomes (Figure 2B, lane
9). The presence of purified CAF-1 in the 293T
extract- and MutL-containing reaction mixture
also caused a significant decrease in the yield of
the ~130-nt product of degradation of the
irreparable O6-mG-containing DNA (Figure 5AB). The effect of CAF-1 on the yield of the
~130-nt product was especially pronounced in
the reaction mixture in which the concentration
of the 3-nicked O6-mG-T DNA was decreased
four times (Figure 5F). In this case, the addition
of purified CAF-1 reduced the yield of the ~130nt degradation product by 5 fold. In the above
experiments, we included 2.4 pmol of purified
CAF-1 in the 293T extract- and MutLcontaining
reaction
mixtures.
Further
experiments showed that the addition of 0.3
pmol of purified CAF-1 was sufficient to cause a
significant decrease in the yield of the ~130-nt

The MMR system and CAF-1

DISCUSSION
Mammalian MGMT-deficient and yeast
mgt1 rad52 cells are efficiently killed by low
methylating
agents
doses
of
Sn1-type
(46,47,78,80-82). Several anticancer therapies
exploit the marked sensitivity of MGMTdeficient cells to Sn1-type methylating agents.
The marked sensitivity of mammalian MGMTdeficient and yeast mgt1 rad52 cells to Sn1type methylating agents is a result of the MMR
system-dependent
cytotoxic
response
(46,47,78,80-82). Previous research showed that
the cytotoxic response to the Sn1-type
methylating drug involves MMR systemdependent degradation of irreparable O6-mGcontaining DNA that leads to the formation of
lethal double-strand breaks (47,53,56). The
MMR system starts to degrade irreparable O6mG-containing nascent DNA behind the
replication fork (53). At the same time, this
DNA is incorporated into nucleosomes in the
CAF-1-orchestrated process (57-61). It was
previously unknown whether concomitant CAF1-dependent nucleosome assembly affects
degradation of irreparable O6-mG-containing
DNA by the MMR system. We have found that
CAF-1 suppresses the activity of the MMR
system in the cytotoxic response of yeast mgt1
rad52 cells to MNNG (Figure 1). We have
also found that in an MGMT-deficient extract
system, CAF-1-dependent incorporation of an
irreparable O6-mG-T mispair-containing DNA
into nucleosomes (Figure 2B) correlates with a
substantial decrease in degradation of this DNA
by
a
MutL
endonuclease-dependent
mechanism (Figures 5, 6, and 7). These findings
imply that CAF-1-dependent packaging of
irreparable O6-mG-T mispair-containing DNA
into nucleosomes suppresses its degradation by
the MMR system, therefore defending the cell
against killing by the Sn1-type methylating drug.
Consistent with this, we have established that
loss of CAF-1 does not affect the sensitivity of
yeast mgt1 rad52 cells to bleomycin,
camptothecin, and hydroxyurea, DNA damaging
drugs that kill cells in an MMR systemindependent manner (Figure 1). It is known that
1-2 double-strand breaks are sufficient to kill the
yeast rad52 cell (83). Therefore, the fact that the
MNNG treatment kills the cac1 mgt1 rad52
and cac2 mgt1 rad52 cells more efficiently
than the mgt1 rad52 cells indicates that CAF1 loss increases the fraction of MNNG-treated
cells that experience at least 1-2 double-strand
breaks.

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MutL endonuclease, the mismatch recognition

factor MutS, the PCNA clamp, the RFC clamp
loader, and the single-stranded DNA-binding
protein RPA.
It can be seen that the
discontinuous strand of the 3-nicked O6-mG-T
DNA was degraded in the defined system in a
MutL endonuclease concentration-dependent
manner (Figure 8A, lanes 13-17, and Figure 8B).
The level of degradation of the discontinuous
strand of the 3 nicked O6-mG-T DNA was 5-6
times higher than that of degradation of the
discontinuous strand of the control 3 nicked A-T
DNA (Figure 8B). No degradation of the
discontinuous strand of the 3 nicked O6-mG-T
DNA was observed in the defined system in
which the endonuclease-deficient MutLE705K substituted for MutL (Figure 8A, lanes
6, 12, and 18, and Figure 8C). This information
indicated that MutL provided the endonuclease
activity that degraded the discontinuous strand
of an irreparable O6-mG-T mispair-containing
DNA in the presence of MutS, PCNA, RFC,
and RPA. Importantly, the ~130-nt fragment that
was detected in the cell extract system (Figure
5A, lane 10) was not a preferred product of the
endonuclease reaction in the defined system
(Figure 8A, lanes 15-17). This is an indication
that the defined system lacks one or more factors
that are involved in the formation of the ~130-nt
fragment in the extract system. While degrading
the discontinuous strand, MutL endonuclease
did not incise continuous strand of the 3 nicked
O6-mG-T DNA (Figure 8E, lanes 13-17). An
inspection of the data in Figure 8A showed that
MutL endonuclease incised the discontinuous
strand of the 3 nicked O6-mG-T DNA at random
sites, displaying no significant site or sequence
specificity. These findings suggested that MutL
endonuclease contributes to MMR systemdependent degradation of irreparable O6-mG-T
mispair-containing DNA by introducing strand
breaks at random sites on the discontinuous
strand. In addition, these findings suggested that
processing of the MutL endonuclease-incised
discontinuous strand of the 3 nicked O6-mG-T
DNA in the extract system led to the formation
of the ~130-nt product (Figure 4A, lane 10). In
agreement with this idea, the pattern of
degradation of the discontinuous strand of the 3
nicked O6-mG-T DNA in the same extract
system that was supplemented with the DNA
polymerase inhibitor aphidicolin (Figure 4A,
lane 14) is similar to that of degradation of the
discontinuous strand of the 3 nicked O6-mG-T
DNA in the defined system (Figure 8A, lanes
13-17).

The MMR system and CAF-1

methylating drug. Although the major product of
persistent
MutL
endonuclease-dependent
degradation of the 3 nicked O6-mG-T DNA in
the extract system has a size of ~130 nt (Figures
4A and 5A, lane 10, and Figure 6A, lane 8), the
products of MutL endonuclease-dependent
degradation of the 3 nicked O6-mG-T DNA in
the extract system that was supplemented with
aphidicolin have sizes in the range of 100-2,000nt (Figure 4A-B, lane 14). Because aphidicolin is
an inhibitor of the biosynthetic activities of Pol
and Pol , these findings suggest that DNA
synthesis and ligation that occur on the MutLdegraded DNA in the extract system in the
absence of aphidicolin are necessary to remove
the majority of the 100-2,000-nt products.
It is important to note that a previous
work
described
that
MutL-dependent
processing of the 3-nicked O6-mG-T DNA in
the HCT116BBR nuclear extract-containing
system leads to the formation of a ~130-nt
product (52) that does not appear to be different
from the one that we have detected in our extract
system (Figures 4-7). This observation implies
that reactions that occur in different extract
systems generate the same product of
degradation of the 3-nicked O6-mG-T DNA. We
have observed that MutL endonucleasedependent degradation of the 3-nicked O6-mG-T
DNA in our extract system leads to the
formation of a gapped product (Figure 7). The
gap was generated in the discontinuous strand in
the presence of dNTPs and was located
downstream from the mispaired T. Thus, the
MMR system-dependent processing of the 3nicked O6-mG-T DNA in the cell extract
generates two kinds of DNA products: one of
them carries the gap (Figure 7) and the other
contains the ~130-nt fragment (Figures 5A and
6A). It is likely that the gap is formed when the
excision step is not blocked by the O6-mG,
whereas the ~130-nt fragment is formed when
the excision step is blocked by the lesion. The
presence of gap in the irreparable DNA is in a
good agreement with a previous study that
documented that gaps are formed behind
replication forks in response to treatment of both
mammalian MGMT-deficient cells and yeast
mgt1 rad52 cells with MNNG (53). The fact
that the gap was generated downstream from the
mismatched T (Figure 7) suggests that O6-mG is
a stronger block for the DNA polymerization
reaction than for the excision reaction.
methylating
drug
The
Sn1-type
temozolomide is used for treatment of
glioblastoma patients. However, recurrent
glioblastomas often arise during post-treatment

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In addition to CAF-1, S. cerevisiae cells

contain several other histone chaperones
including HIR and Rtt106 (75). Our results
indicated that loss of HIR2 or RTT106 does not
increase the sensitivity of mgt1 rad52 cells to
MNNG (Figure 1G). Thus, these results imply
that neither of these histone chaperones has a
non-redundant function that provides a
protection for mgt1 rad52 cells from the
cytotoxic activity of the MMR system. We
determined that decreasing histone H3-H4 gene
dosage by deletion of the HHT2-HHF2 locus in
the mgt1 rad52 cells does not change their
sensitivity to MNNG (Figure 1G). A previous
report described that hht2-hhf2 does not affect
the level of the chromatin H3-H4 histones but
decreases the level of the soluble H3-H4
histones two fold (84). Thus, it appears that a
small decrease in the level of the soluble
histones H3-H4 does not affect the cytotoxic
activity of the MMR system.
An earlier study has implicated MutL
endonuclease activity in the cytotoxic response
to the Sn1-type methylating drug in the yeast and
mammalian cells (68). We have now shown that
activated MutL endonuclease degrades the
discontinuous strand of an irreparable O6-mG-T
mispair-containing DNA in an extract system
and in a purified system (Figures 4-8). This
information implies that MutL endonucleasedependent degradation of the discontinuous
strand of irreparable O6-mG-T mispaircontaining nuclear DNA is involved in the
cytotoxic response to the Sn1-type methylating
drug. MutL endonuclease degrades the 3nicked O6-mG-T and G-T DNA substrates in the
presence of MutS, PCNA, RFC, and RPA in a
very similar way (Figure 8A, lanes 3-5 and lanes
15-17), suggesting that the same mechanism
activates MutL endonuclease in MMR and in
the cytotoxic response to the Sn1-type
methylating drug. The mechanism of activation
of MutL endonuclease in MMR requires the
presence of MutS and a mismatch (37,39,42)
(Figure 8). The O6-mG-T mispair is one of many
mispairs recognized by MutS (21). A
crystallographic study determined that MutS
has the same structure in the MutS-G-T DNA
and MutS-O6-mG-T DNA crystals (85). This
information and the results of our analysis of the
defined reactions (Figure 8) suggest that
adoption of the same structure by MutS on the
G-T mispair and on the irreparable O6-mG-T
mispair permits the protein to convey the same
activating signal to MutL endonuclease during
MMR and the cytotoxic response to the Sn1-type

The MMR system and CAF-1

period (86). This indicates that an approach that
increases the sensitivity of MGMT-deficient
tumors to treatment with the Sn1-type
methylating drug might suppress recurrent
cancers. More research is needed to determine
whether
defective
replication-coupled
nucleosome assembly increases the sensitivity of
MGMT-deficient cancer cells to treatment with
the Sn1-type methylating drug.
EXPERIMENTAL PROCEDURES
Yeast Strains S. cerevisiae strains that
were used in this study were derivatives of the
wild-type haploid strain E134 (MAT ade5-1
lys2::InsE-A14 trp1-289 his7-2 leu2-3,112 ura352) (13). The lithium acetate/PEG4000/DMSO
transformation method and PCR-amplified
disruption cassettes were used to generate the
strains.
MNNG and Bleomycin Cytotoxicity
Assays Liquid YPDAU medium (1% yeast
extract, 2% bacto-peptone, 2% dextrose, 60
mg/L adenine, 60 mg/L uracil), YPDAU plates,
1-mM stock solutions of MNNG (Wako
Chemicals USA) in DMSO, a 2 mg/ml stock
solution
of
bleomycin
(Santa
Cruz
Biotechnology) in DMSO, and DMSO were
used in the assays. Yeast cultures were grown to
saturation in liquid YPDAU for ~20 h at 30C.
The saturated cultures were diluted 10-fold in
fresh medium and grown for 4 h at 30C. The
cultures were then diluted with fresh medium to
OD600=1.3, and aliquots of the diluted cultures
were treated with 1 M MNNG, 0.1% DMSO
(vehicle control in the MNNG toxicity assay), 30
g/ml bleomycin, and 1.5% DMSO (vehicle
control in the bleomycin cytotoxicity assay) for
2 h at 30C. After treatment, the cultures were
diluted, and appropriate dilutions of the cultures
were spread on YPDAU plates. The pates were
incubated for 3-4 days at 30C, and colonies
were counted. A somewhat different MNNG
cytotoxicity assay was also used in this study. In
this assay, ten-fold serial dilutions of the treated
cultures were made and spotted on YPDAU
plates. The plates were incubated for 2 days at
30C and photographed.
Camptothecin
and
Hydroxyurea
Cytotoxicity Assays Yeast cultures were grown
to saturation as described above and diluted to
OD600=1.4 with sterile water. Ten-fold serial
dilutions of the cultures were prepared and
spotted on YPDAU plates, YPDAU plates
containing 0.5 g/ml camptothecin (Enzo Life
Science), and YPDAU plates containing 10 mM
hydroxyurea (US Biological). After incubation

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for 2 days at 30C, the plates were

photographed.
Cell Extract and Recombinant Proteins
293T cells were grown as an attached culture
in DMEM/high glucose medium that was
supplemented with 10% FBS, 100 U/ml
penicillin, 100 g/ml streptomycin, and 0.29
g/ml L-glutamine. Cytosolic and nuclear
extracts from proliferating 293T cells were
prepared according to a described procedure
(63). Recombinant human CAF-1, MutS,
MutL, MutL-E705K, PCNA, RFC, and RPA
were isolated in near homogeneous forms as
previously described (37,63).
Western Blotting Samples of the
purified CAF-1, 293T cytosolic extract (15 g),
and 293T nuclear extract (15 g) were separated
on a denaturing SDS gel and transferred onto a
PVDF membrane. After the protein transfer step,
the membrane was incubated with -CAF-1
p150 antibodies (cat. no. sc-10772, lot E2004,
rabbit polyclonal IgG, SantaCruz) and then with
ECL HRP conjugated secondary antibodies (cat.
no. NA934V, lot 389592, donkey antibody, GE
HealthCare).
Immune
complexes
were
visualized utilizing ECL2 western blotting
substrate (ThermoFisher) and a CCD camera.
Amounts of CAF-1 in the samples of the 293T
cytosolic and nuclear extracts were measured by
quantification of the data with the ImageJ
software.
DNA substrates, Oligonucleotides, and
32
P-Labeled Hybridization Probes 3-nicked
O6-mG-T, 3-nicked G-T, and 3-nicked A-T
DNAs were essentially prepared as previously
described (52), except that after DNA ligation,
unligated
material
was
degraded
by
Exounuclease III (NEB) and the remaining DNA
was purified by chromatography on BNDcellulose (Sigma). The O6-mG-containing
oligonucleotide that was used to prepare the 3nicked O6-mG-T DNA was synthesized by
Midland Certified Reagent Company. All other
oligonucleotides used in this study were
synthesized by IDT. To prepare a 32P-labeled
hybridization probe, the oligonucleotide was
labeled at the 5 end with 32P by T4
polynucleotide kinase in the presence of [32
P]ATP. The sequences of oligonucleotides
used to construct the DNA substrates and
prepare 32P-labeled hybridization probes are
shown in Table 1.
Nucleosome Assembly Reactions in
293T Cytosolic Extract-containing Mixtures
The nucleosome assembly reactions were carried
out at 37C in 40-l mixtures that contained 20
mM HEPES-NaOH (pH 7.4), 100 mM KCl, 8

The MMR system and CAF-1

Acknowledgements: We thank Farid F. Kadyrov for critical reading of the manuscript.

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mispairs occurred in a reaction mixture, a

fraction of the recovered DNA was cleaved with
BanI and XhoI and separated on a 1.2% agarose
gel, followed by staining of the gel with
ethidium bromide and quantification of DNA
species with the ImageJ software. To detect
whether a 3 nicked DNA was degraded in the
reaction, a fraction of the recovered DNA was
cleaved with ClaI, separated on an alkaline 1.4%
agarose gel, transferred onto a nylon membrane
and hybridized with a 32P-labeled probe. To
detect whether gapped DNA was produced in a
reaction mixture, a fraction of the recovered
DNA was digested with AhdI, resolved on an
alkaline 1.4% agarose gel, transferred onto a
nylon membrane and hybridized with an
indicated 32P-labeled probe. Indirectly labeled
DNA species were visualized and quantified as
described above.
Cleavage of DNA by Activated MutL
Endonuclease in a Defined System The
reactions were performed in 40-l mixtures that
contained 20 mM HEPES-NaOH (pH 7.4), 120
mM KCl, 5 mM MgCl2, 3 mM ATP, 2 mM
DTT, 0.2 mg/ml BSA, 2% glycerol (v/v), and 2
nM (81 fmol) of a 3-nicked DNA (the 3-nicked
O6-mG-T DNA, the 3-nicked G-T DNA, or the
3-nicked A-T DNA). When indicated, MutS
(40 nM), MutL (2, 6, or 20 nM), MutLE705K (20 nM), PCNA (24 nM), RFC (4 nM),
and RPA (40 nM) were included in the reaction
mixtures. The reaction mixtures were incubated
for 10 minutes at 37C, and each reaction was
terminated by the addition of a 30-l mixture
containing 0.35% SDS, 0.4 M NaCl, 13 mM
EDTA, 0.33 mg/ml Proteinase K, and 2 mg/ml
glycogen, followed by incubation of the
mixtures for 15 min at 50C. The mixtures were
then extracted by phenol/chloroform, and the
DNAs were recovered by isopropanol
precipitation. Recovered DNAs that were
digested or not with ClaI were separated on
alkaline 1.4% denaturing agarose gels,
transferred onto nylon membranes, and
hybridized with 32P-labeled probes. Indirectly
labeled DNA species were visualized and
quantified as described above.

mM MgCl2, 2 mM DTT, 0.2 mg/ml BSA, 0.1

mM each of the four dNTPs, 3 mM ATP, 20
mM creatine phosphate, 0.02 mg/ml creatine
phosphokinase, 1% glycerol (v/v), 75 g 293T
cytosolic extract, purified CAF-1 (0 or 1.2
pmol), MutL (0 or 1.6 pmol) and 81 fmol (0.1
g) of a 3-nicked DNA (the 3-nicked O6-mG-T
DNA, the 3-nicked G-T DNA, or the 3-nicked
A-T DNA). After 60 minutes of incubation, 35l fraction of each reaction mixture was mixed
with a 5-l mixture containing 20 mM HEPESNaOH (pH 7.4), 64 mM CaCl2, 8 U/l
micrococcal nuclease and .02 mg/ml RNAseA
and incubated for 20 min at 21-23C. Each
reaction mixture was then supplemented with a
10-l solution containing 0.5% SDS, 150 mM
EDTA, 40% glycerol, and 2 mg/ml proteinase K,
followed by incubation of the mixture for 20 min
at 50C. Proteinase K in the mixtures was
inactivated by the addition of PMSF to a final
concentration of 0.8 mM. The nucleosome
assembly products were separated on native
1.7%
agarose
gels,
transferred
onto
nitrocellulose membranes and hybridized with
32
P-labeled probe c154. Indirectly labeled DNA
species were visualized with a Typhoon
biomolecular imager (GE HealthCare) and
quantified with an ImageQuant software.
Mismatch-Provoked Reactions in an
extract system The mismatch-provoked and
control reactions in the extract system were
carried out at 37C in 80-l mixtures containing
20 mM HEPES-NaOH (pH 7.4), 100 mM KCl, 8
mM MgCl2, 2 mM DTT, 0.2 mg/ml BSA, 0.1
mM each of the four dNTPs, 3 mM ATP, 20
mM creatine phosphate, 0.02 mg/ml creatine
phosphokinase, 1% glycerol (v/v), 150 g 293T
cell extract, MutL (0 or 3.2 pmol), MutLE705K (0 or 3.2 pmol), purified CAF-1 (0, 0.3,
0.6, or 2.4 pmol) and 41 or 162 fmol of a 3
nicked DNA (the 3-nicked O6-mG-T DNA, the
3-nicked G-T DNA, or the 3-nicked A-T
DNA). The reaction mixtures were incubated for
10-120 min. Reactions in each mixture were
stopped by the addition of a 60-l solution
containing 0.35% SDS, 0.4 M NaCl, 13 mM
EDTA, 0.33 mg/ml Proteinase K, and 1 mg/ml
glycogen, and the resulting mixtures were
incubated for 20 min at 50C, followed by their
extraction with phenol/chloroform mixture.
DNAs present in the supernatants were
recovered by isopropanol precipitation. To
detect whether the repair of O6-mG-T or G-T

The MMR system and CAF-1

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of
this article.
FOOTNOTES
Research reported in this publication was supported by the National Institute of General Medical
Sciences of the National Institutes of Health under Award Number R01GM095758. The content is
solely the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health.
Author contributions: LK, BD, and FK performed experiments and analyzed data. FK and LK
designed experiments, contributed reagents, prepared the figures, and wrote the paper.
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R., Barrett, J. C., and Kunkel, T. A. (1997) Correction of hypermutability, N-methyl-N'-nitroN-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing
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579-592
Cahill, D. P., Levine, K. K., Betensky, R. A., Codd, P. J., Romany, C. A., Reavie, L. B.,
Batchelor, T. T., Futreal, P. A., Stratton, M. R., Curry, W. T., Iafrate, A. J., and Louis, D. N.
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The MMR system and CAF-1

TABLES
TABLE 1. Oligonucleotides that were used to prepare the DNA substrates and
hybridization probes
Oligonucleotide1
Oligonucleotide sequence

32

5-GCTACCGTCCTCGAAGCTTCCGCATCGGAGTCGACG-3

AH1A G-T

5-GCTACCGTCCTCGAGGCTTCCGCATCGGAGTCGACG-3

AH1A O6-mG-T

5-GCTACCGTCCTCGAO6mGGCTTCCGCATCGGAGTCGACG-3

ts1

5-CTCTTCCTTTTTCAATTGGGATCC-3

ts154

5-TCGAGTCTCTCGCTACCGTCCTCG-3

ts182

5-TTCCGCATCGGAGTCGACGGCTTC-3

ts1131

5-GACAGTTACCAATGCTTAATCAGTG-3

c29

5-CTGAGGATCCCAATTGAAAAAGGA-3

c154

5-CGAGGACGGTAGCGAGAGACTCGA-3

, the first three oligonucleotides were used to prepare the 3-nicked DNA substrates, and the
remaining oligonucleotides were used to prepare 32P-labeled hybridization probes.

15

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AH1A A-T

P-labeled

The MMR system and CAF-1

FIGURE LEGENDS
FIGURE 1. CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast
mgt1 rad52 cells to MNNG. Cytotoxicity assays were carried out as detailed under
EXPERIMENTAL PROCEDURES. A and B, cytotoxic responses of cac1 mgt1 rad52 and cac2
mgt1 rad52 cells to treatment with 1 M MNNG. 10-fold serial dilutions of yeast cultures that
were treated or not treated with 1 M MNNG were spotted on the YPDAU plates. C, quantitative
analysis of cytotoxic responses of cac1 mgt1 rad52 and cac2 mgt1 rad52 cells to treatment
with 1 M MNNG (D) quantitative analysis of cytotoxic response of cac1 mgt1 rad52 to
treatment 30 g/ml bleomycin. The data in C and D are averages 1 S.D. (n 3). E and F, cytotoxic
responses of cac1 mgt1 rad52 and cac2 mgt1 rad52 cells to treatments with 0.5 g/ml
camptothecin and 10 mM HU. 10-fold serial dilutions of yeast cultures were spotted on the YPDAU
plates, YPDAU plate with 0.5 g/ml camptothecin, and YPDAU plate with 10 mM HU. G, cytotoxic
responses of hht2-hhf2 mgt1 rad52, hir2 mgt1 rad52, and rtt106 mgt1 rad52 cells to
treatment with 1 M MNNG. 10-fold serial dilutions of yeast cultures that were treated or not with 1
M MNNG were spotted on the YPDAU plates.

FIGURE 3. MMR system reconstituted with an extract and purified MutL does not correct a
mispair that contains O6-mG on the continuous strand. The experiments were carried out and
analyzed as described under EXPERIMENTAL PROCEDURES. Each reaction mixture contained a
DNA substrate (162 fmol) and the other indicated components and was incubated for 10 min at 37C.
No exogenous dNTPs were present in the reaction mixtures that included aphidicolin (0.1 mM). To
score MMR, the recovered DNAs were digested with XhoI and BanI, separated on native agarose
gels, and visualized with ethidium bromide staining. A, outline of the assay. The assay is based on the
knowledge that the repair of the mismatched T on the 3-nicked G-T or the 3-nicked O6-mG-T DNA
restores the XhoI site. B, DNA species present in the indicated reaction mixtures. C, amounts of
MMR products formed in the indicated reaction mixtures. The data are averages 1 S.D. (n = 3) and
were obtained by quantification of images including the one shown in B.
FIGURE 4. MMR system reconstituted with an extract and purified MutL degrades the
discontinuous strand of an irreparable O6-mG-T mispair-containing DNA. The experiments were
carried out as described in Figure 3. The recovered DNAs were digested with ClaI, separated on
alkaline agarose gels, transferred onto nylon membranes, and hybridized with the 32P-labeled probes
ts154 and ts1 that are complementary to the discontinuous strands. A and B, DNA species visualized
with the ts154 probe and the ts1 probe, respectively. Each of the diagrams outlines relative positions
of the probe (a bar with an asterisk), the unique ClaI site, the strand break, and a mismatch. The
distance between the ClaI site and the mismatch is 145 bp.
FIGURE 5. CAF-1-dependent suppression of persistent MutL endonuclease-dependent
degradation of an irreparable O6-mG-T mispair-containing DNA in an extract system. The
experiments were carried out and analyzed as described under EXPERIMENTAL PROCEDURES.
The reaction mixtures included the indicated components and were incubated for 60 min (A, C-F) or
10-120 min (B) at 37C. Each reaction mixture analyzed in A-E contained 162 fmol of a DNA
substrate, and each reaction mixture analyzed in F contained 41 fmol of a DNA substrate. Exogenous
dNTPs were not included in the reaction mixtures that contained aphidicolin (0.1 mM). The recovered
DNAs were digested with ClaI, separated on alkaline agarose gels, transferred onto nylon membranes,
and hybridized with the 32P-labeled probes ts154, ts182, ts1, and c154. The ts154, ts182, and ts1
probes are complementary to the discontinuous strands, and the c154 probe is complementary to the
continuous strands. A, DNA species visualized with the 32P-labeled probe ts154. B, effect of the

16

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FIGURE 2. Nucleosome assembly reactions reconstituted with an extract and purified CAF-1.
The experiments were conducted and analyzed as described under EXPERIMENTAL
PROCEDURES. A, analysis of CAF-1 in 293T cytosolic extract (293T CE) and 293T nuclear extract
(293T NE) by Western blot with antibodies against the CAF-1 p150 subunit. Quantification of the
data showed that 293T cytosolic extract (150 g) contains 0.077 0.009 pmol of CAF-1. B, analysis
of nucleosome assembly in reaction mixtures containing the indicated components. The nucleosome
assembly products were visualized with the 32P-labeled probe c154 that is complementary to the
continuous strand. A diagram on the left indicates the relative positions of the 32P-labeled probe (a bar
with an asterisk), the strand break, and a mismatch.

The MMR system and CAF-1

presence of purified CAF-1 (2.4 pmol) on the formation of the ~130-nt product of degradation of the
discontinuous strand of the 3-nicked O6-mG-T DNA. The data were obtained by quantification of
images including those shown in A and Figure 4A and are averages 1 S.D. (n 2). C, D, and E,
DNA species visualized with the 32P-labeled probes ts182, ts1, and c154, respectively. Each of the
diagrams outlines relative positions of the probe (a bar with an asterisk), the unique ClaI site, the
strand break, and a mismatch. F, quantification of ~130-nt degradation products formed in the
indicated reaction mixtures. The data were obtained using the ts154 probe and are averages 1 S.D.
(n=3).

FIGURE 7. CAF-1-dependent suppression of the formation of a gap in an irreparable O6-mG-T

mispair-containing DNA in an extract system. The experiments were performed and analyzed as
described under EXPERIMENTAL PROCEDURES. Each reaction mixture contained a DNA
substrate (41 fmol) and other indicated components and was incubated for 60 min at 37C. The
recovered DNA products were digested with AhdI, separated on native agarose gels, transferred onto
nylon membranes and hybridized with the 32P-labeled probes c154 and ts154. A, outline of the assay.
B and C, DNA species visualized with the 32P-labeled probes c154 and ts154, respectively. The c154
probe is complementary to the continuous strand and the ts154 probe is complementary to the
discontinuous strand. Each diagram depicts the relative positions of the 32P-labeled probe (a bar with
an asterisk), AhdI sites, the strand break, and a mismatch. D, quantification of gapped DNA formed in
MutL endonuclease-dependent reactions in the indicated reaction mixtures. E, effects of the different
amounts of purified CAF-1 on the formation of the gapped product of the 3-nicked O6-mG-T DNA in
293T cytosolic extract-containing reaction mixture. The reaction mixtures contained 293T cytosolic
extract (150 g), MutL (3.2 pmol), and CAF-1 (0, 0.3, 0.6, or 2.4 pmol). The data in D and E were
obtained with the 32P-labeled probe c154 and are averages 1 S.D. (n = 3).
FIGURE 8. MutL endonuclease-dependent degradation of an irreparable O6-mG-T mispaircontaining DNA in a defined system. The experiments were performed and analyzed as described
under EXPERIMENTAL PROCEDURES. Each reaction mixture contained a DNA substrate (81
fmol) and the indicated proteins, and was incubated for 10 min at 37C. When MutS, PCNA, RFC,
and RPA were present in the reaction mixtures, their concentrations were 40 nM, 24 nM, 4 nM, and
40 nM, respectively. The recovered DNAs that either were not digested (A and C) or were digested
(E) with ClaI were resolved on alkaline agarose gels, and hybridized with the 32P-labeled probes
ts154, ts1, and c29. The ts154 and ts1 probes are complementary the discontinuous strands, and the
c29 probe is complementary to the continuous strands. A and C, DNA species visualized with the 32Plabeled probes ts154 and ts1, respectively. B, degradation of the discontinuous strands of the 3 nicked
DNA substrates as a function of MutL endonuclease concentration. The data were obtained by
quantification of images generated with the 32P-labeled probe ts154. One of the images is shown in A.
D, degradation of the discontinuous strands of the 3 nicked DNA substrates in the presence of the
MutL-E705K mutant. The data were obtained by quantification of images generated with the 32Plabeled probe ts1. One of the images is shown in C. The data in B and D are averages 1 S.D. (n = 3).
E, DNA species visualized with the 32P-labeled probe c29. Each of the diagrams depicts the relative
positions of the 32P-labeled probe (a bar with an asterisk), the strand break, and a mismatch.

17

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FIGURE 6. CAF-1 suppresses persistent degradation of an irreparable O6-mG-T mispaircontaining DNA in an extract system in a concentration-dependent manner. The experiments
were carried out and analyzed as detailed under EXPERIMENTAL PROCEDURES. Each reaction
mixture contained a DNA substrate (41 fmol) and the other indicated components and was incubated
for 60 min at 37C. The recovered DNAs were digested with ClaI, separated on an alkaline agarose
gel, transferred onto a nylon membrane, and hybridized with the 32P-labeled probe ts154 that is
complementary to the discontinuous strand. A, DNA species visualized in one of the experiments. The
diagram outlines relative positions of the probe (a bar with an asterisk), unique ClaI site, strand break,
and mismatch. B, formation of the ~130-nt product of degradation of the 3-nicked O6-mG-T DNA as
a function of amount of purified CAF-1. The data were obtained by quantification of images including
the one in A and are averages 1 S.D. (n = 3).

MNNG
none

Figure 1

1 M

MNNG
none

WT

mgt1 rad52

mgt1 rad52

msh2 mgt1 rad52

mlh1 mgt1 rad52

1 M

cac2 mgt1 rad52

cac1 mgt1

cac2 msh2 mgt1 rad52

cac1 mgt1 rad52

cac2 mgt1

cac1 mlh1 mgt1 rad52

mgt1
WT

115%

89%

72% 71%

47% 46%

35

10
1.7%

0.4%
0.2%

0.1

24%

30
23%

25
20
15
10

3.8%

2.6% 3.2%

c1

ca

d5

gt

gt

ra

d5

ra
m

c1

ca

ca

c1

ca

lh

c1

gt

gt

gt

ild

ra

d5

ty
pe

ild
ty
1
pe

m
r
m
gt ad
lh
1 52
1

r
m
m
lh
gt ad5
1 1
2
m
r
gt ad
1 52
m ra
gt d5
1 2
ca
ca
c2
c2
ca

c1
m
m

sh
gt
2 1
m
r
sh mg ad
t
2 1 52

m
r
gt ad
5
1
2
ra
d5
2

E
WT
mgt1 rad52
mlh1 mgt1 rad52
cac1 mgt1 rad52
cac1 mlh1 mgt1 rad52
cac1 mgt1

Camptothecin
none

Hydroxyurea

0.5 g/ml

none

10 mM

Camptothecin

mgt1 rad52
msh2 mgt1 rad52
cac2 mgt1 rad52
cac2 msh2 mgt1 rad52
cac2 mgt1
mgt1
WT

MNNG

WT
mgt1 rad52
cac1 mgt1 rad52
hht2-hhf2 mgt1 rad52
hir2 mgt1 rad52
rtt106 mgt1 rad52

18

none

1 M

none

0.5 g/ml

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

Survival (%), 1 M MNNG

100

Survival (%), 30 g/ml BLE

Hydroxyurea
none

10 mM

29
3
29 T C
3T E
N
E

Figure 2
3 G-T
MutL
CAF-1
293T CE

- CAF-1

191 -

3 O -mG-T

3 A-T

- - ++ - - - ++ - - - ++ - - - ++ - - - ++ - - - ++
- ++++ - ++++ - ++++

564 bp -

- trinucleosome
- dinucleosome
- mononucleosome

125 bp -

28 -

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

Lane

Figure 3

B
+ 293T cytosolic extract, Mut ,
BanI

Mg 2+ ,

Repair

No repair

ATP, and dNTPs

+
+

+
+
+

+
+

+
+

+
+
+

3 O6-mG-T

+
+

+
+
+

+
+

+
+

3 A-T

+
+
+

+
+

not repaired DNA -

XhoI

repaired DNA Lane

BanI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

C
70

DNA repair (fmol)

60
50
40
30
20
10
0

MutL
CAF-1 (2.4 pmol)
MutL-E705K
293T CE

+
+

+
+
+

+
+

+
+

3 G-T

20

+
+

+
+
+

+
+

3 O6-mG-T

+
+

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

BanI

MutL
CAF-1 (2.4 pmol)
MutL-E705K
Aphidicolin
293T CE

3 G-T

Figure 4

A
MutL
CAF-1 (2.4 pmol)
MutL-E705K
Aphidicolin
293T CE
2,000-

+
+

+
+
+

+
+

-+
- +-+
++

+
+

+
+
+

+
+

+
+

3 A-T

+
+
+

ClaI

560-

Lane

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

+
+

+
+
+

+
+

-+
- +-+
++

+
+

+
+
+

+
+

+
+

+
+
+

+
+

2,000-

560-

130Lane

21

3 A-T

3 O -mG-T

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

~130-nt
product

130-

3 G-T

MutL
CAF-1 (2.4 pmol)
MutL-E705K
Aphidicolin
293T CE

+
+

ClaI

3 O -mG-T

3 G-T

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

MutL
CAF-1 (2.4 pmol)
MutL-E705K
Aphidicolin
293T CE

+
+

+
+
+

+
+

-+
- +-+
++

3 A-T

3 O -mG-T

+
+

+
+
+

+
+

+
+

+
+
+

+
+

2,000-

560~130-nt
product

1301 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

C
MutL
CAF-1 (2.4 pmol)
MutL-E705K
Aphidicolin
293T CE

ClaI

3 G-T

+
+

+
+
+

+
+

-+
- +-+
++

+
+

+
+
+

+
+

+
+

+
+
+

+
+

560-

130-

130-

MutL
CAF-1 (2.4 pmol)
MutL-E705K
Aphidicolin
293T CE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

3 O -mG-T

3 G-T

2,000-

+
+

+
+
+

+
+

-+
- +-+
++

+
+

+
+
+

+
+

+
+

Lane

3 A-T

+
+
+

+
+

560-

+ purified CAF-1

4
2
0

20 40 60 80 100 120 140

Time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

22

3 O -mG-T

+
+

+
+
+

+
+

-+
- +-+
++

+
+

+
+
+

+
+

+
+

3 A-T

+
+
+

+
+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

4
3
2
1

0
MutL
CAF-1 (2.4 pmol)
MutL-E705K
293T CE

130Lane

2,000-

ClaI

560-

no purified CAF-1

3 G-T

Lane

3 O -mG-T

MutL
CAF-1 (2.4 pmol)
MutL-E705K
Aphidicolin
293T CE

2,000-

ClaI

10

3 A-T

3 O -mG-T

Figure 5

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

Lane

~130-nt product of degradation

of the discontinuous strands (fmol)

ClaI

3 G-T

~130-nt product of MutL-dependent

degradation of the discontinuous strand (fmol)

+
+

+
+
+

3 G-T

+
+

+
+

+
+

+
+
+
6

+
+

+
+

3 O -mG-T

+
+

3 A-T

Figure 6

B
0.6
0.3

0.6
0.3

- - - - - - +++ - - +++
- + +++ - + +++

purified CAF-1 (pmol)

MutL
293T CE
2000ClaI

3 O -mG-T

~130-nt product of degradation

of the discontinuous strand of
6
3 O -mG-T DNA (fmol)

3 G-T

560~130-nt
product

1301 2 3 4 5 6 7 8 9 10

23

2.5
2
1.5
1
0.5
0

0.4 0.8 1.2 1.6 2.0 2.4 2.8

purified CAF-1 (pmol)

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

Lane

Figure 7

B
MutL
CAF-1 (2.4 pmol)
MutL-E705K
293T CE

AhdI
3

+ 293T cytosolic extract, MutL,

Mg 2+ , ATP, and dNTPs

AhdI

3 O6-mG-T
- + + - - - + + - - - - +
++ + ++

3 G-T
++ - - ++ - - - +
++++

3 A-T
- +
- - ++
] gapped DNA

2000-

AhdI
AhdI

560-

Lane 1 2 3 4 5 6 7 8 9 10 1112

2
1
0

+ +
- +
+ +

3 O -mG-T

5
4
3
2
1
0

-T
6

0.4 0.8 1.2 1.6 2.0 2.4 2.8

purified CAF-1 (pmol)

24

+ + + MutL
- + - CAF-1 (2.4 pmol)
+ + + 293T CE
G

G
3

-T

Lane 1 2 3 4 5 6 7 8 9 10 1112

-T

560-

MutL-dependent gap formation (fmol)

AhdI

] gapped DNA

2000-

3 A-T
- +
- - ++

AhdI

3 O6-mG-T
- + + - - - + + - - - - +
++ + ++

-m

MutL
CAF-1 (2.4 pmol)
MutL-E705K
293T CE

3 G-T
++ - - ++ - - - +
++++

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

MutL-dependent gap formation (fmol)

AhdI

Figure 8
3 G-T

3 A-T

3 O -mG-T

MutL (nM) 0 0 2 6 20 0 0 0 2 6 20 0 0 0 2 6 20 0
MutL-E705K (nM) 0 0 0 0 0 20 0 0 0 0 0 20 0 0 0 0 0 20
PCNA, RFC, RPA, MutS - + + + + + - + + + + + - + + + + +
2,000-

degradation products

560-

130-

3 G-T

50
6

3 O -mG-T

40
30
20

3 A-T

10
0

MutL (nM) 0 0 2 6 20 0 0 0 2 6 20 0 0 0 2 6 20 0
MutL-E705K (nM) 0 0 0 0 0 20 0 0 0 0 0 20 0 0 0 0 0 20
PCNA, RFC, RPA, MutS - + + + + + - + + + + + - + + + + +
2,000-

130-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

3 G-T

70
60
50
40
30
20
10
0

degradation products

560-

20 nM
MutL
3 A-T

3 O -mG-T

MutL (nM) 0 0 2 6 20 0 0 0 2 6 20 0 0 0 2 6 20 0
MutL-E705K (nM) 0 0 0 0 0 20 0 0 0 0 0 20 0 0 0 0 0 20
PCNA, RFC, RPA, MutS

- +++++ - +++++ - +++++

2,000-

ClaI
560-

130-

Lane

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718

25

20

G
3 -T
O6 A
-m -T
G
-T
3
G
3 3 -T
O 6 A-m T
G
-T

3 O -mG-T

10
15
MutL (nM)

3 A-T

Discontinuous strand degradation (fmol)

3 G-T

Lane

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718

70

20 nM
MutL-E705K

25

Downloaded from http://www.jbc.org/ at BIBLIO DE LA SANTE on December 16, 2016

Lane

Discontinuous strand degradation (fmol)

The major replicative histone chaperone CAF-1 suppresses the activity of the DNA
mismatch repair system in the cytotoxic response to a DNA methylating agent
Lyudmila Y Kadyrova, Basanta K Dahal and Farid A Kadyrov
J. Biol. Chem. published online November 21, 2016

Access the most updated version of this article at doi: 10.1074/jbc.M116.760561

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