4808–4817 Nucleic Acids Research, 2001, Vol. 29, No. 23
© 2001 Oxford University Press
Replication protein A is sequentially phosphorylated
during meiosis
George S. Brush*, Dawn M. Clifford, Suzanne M. Marinco and Amy J. Bartrand
Program in Molecular Biology and Genetics, Karmanos Cancer Institute, Wayne State University,
110 East Warren Avenue, Detroit, MI 48201, USA
Received August 13, 2001; Revised and Accepted October 5, 2001
ABSTRACT
Phosphorylation of the cellular single-stranded DNAbinding protein, replication protein A (RPA), occurs
during normal mitotic cell cycle progression and also
in response to genotoxic stress. In budding yeast,
these reactions require the ATM homolog Mec1, a
central regulator of the DNA replication and DNA
damage checkpoint responses. We now demonstrate
that the middle subunit of yeast RPA (Rfa2) becomes
phosphorylated in two discrete steps during meiosis.
Primary Rfa2 phosphorylation occurs early in
meiotic progression and is independent of DNA
replication, recombination and Mec1. In contrast,
secondary Rfa2 phosphorylation is activated upon
initiation of recombination and requires Mec1. While
the primary Rfa2 phosphoisomer is detectable throughout most of meiosis, the secondary Rfa2 phosphoisomer is only transiently generated and begins to
disappear soon after recombination is complete.
Extensive secondary Rfa2 phosphorylation is observed
in a recombination mutant defective for the pachytene
checkpoint, indicating that Mec1-dependent Rfa2 phosphorylation does not function to maintain meiotic delay
in response to DNA double-strand breaks. Our results
suggest that Mec1-dependent RPA phosphorylation
could be involved in regulating recombination rather
than cell cycle or meiotic progression.
INTRODUCTION
Replication protein A (RPA) is an evolutionarily conserved
single-stranded DNA (ssDNA)-binding protein that is required
for DNA replication, repair and recombination in eukaryotic
cells (1,2). The RPA complex consists of three polypeptides
with molecular weights of ∼70, ∼30 and ∼14 kDa, and the
major ssDNA-binding activity of the protein resides in the
largest of these subunits. While stabilization of ssDNA is
central to its function, RPA also directly associates with other
proteins that are involved in DNA replication, repair and
recombination. These protein–protein interactions are thought
to play a critical role in the initiation of DNA transactions,
suggesting that RPA is an important regulatory target. In
support of this hypothesis, the middle subunit of RPA becomes
phosphorylated during progression through the mitotic cell
cycle and also in response to genotoxic stress (3–7). While the
effect of these post-translational modifications remains
unclear, their conservation from yeast to humans provides
strong evidence that RPA phosphorylation has a significant
impact on the fitness of the eukaryotic cell.
One approach to understanding the function of RPA phosphorylation is to identify and characterize the RPA kinases that
function in vivo. Our previous studies in the budding yeast
Saccharomyces cerevisiae revealed that the protein kinase
Mec1 is required for cell cycle-regulated phosphorylation of
the RPA middle subunit (Rfa2) (7), which occurs during the
S and G2 phases (3). We also demonstrated that various forms
of genotoxic stress lead to Mec1-dependent phosphorylation of
both Rfa2 (7) and the RPA large subunit (Rfa1) (8). Mec1 is a
key regulator of the ‘checkpoint’ responses that function to
delay cell cycle and meiotic progression in response to cellular
perturbations (9–13). Several lines of evidence indicate that
Mec1 is also involved in recombination. During mitotic
growth, mec1 mutants exhibit increased spontaneous intergenic recombination (14), decreased spontaneous and DNA
damage-induced intragenic recombination (15) and decreased
non-homologous end joining (16). During meiosis, mec1
mutants exhibit diminished homologous recombination
(14,17) and enhanced ectopic recombination (17). Functional
Mec1 is also required for redistribution of Sir3 from telomeres
to DNA double-strand breaks (18,19), which are repaired by
recombination (20), and for phosphorylation of the recombination proteins Rad55 and Srs2 (15,21). Finally, Mec1 directs
phosphorylation of histone H2A in response to agents that
cause DNA double-strand breaks, and mutation of the H2A
phosphorylation site confers a defect in non-homologous end
joining without affecting Mec1-mediated cell cycle delay (22).
Mec1 is structurally and functionally related to ATM, the
protein kinase mutated in the pleiotropic human disorder ataxia
telangiectasia (AT) (23). This disease is characterized by
cerebellar degeneration, ocular and facial telangiectasia,
immunodeficiency, infertility, premature aging and an
increased risk of leukemias and lymphomas (24). AT cells are
hypersensitive to killing by ionizing radiation (25) and, like
mec1 mutants, exhibit defects in both checkpoint-mediated cell
cycle delay (26–28) and recombination (29–31). AT cells are
also deficient in IR-induced phosphorylation of several proteins
(32), including RPA (5). Therefore, an evolutionarily conserved
pathway involving Mec1 in yeast and ATM in humans functions
to modify RPA in response to genotoxic stress.
Because Mec1 and ATM are required for normal checkpoint
function, it is possible that RPA phosphorylation serves as a
*To whom correspondence should be addressed. Tel: +1 313 833 0715; Fax: +1 313 832 7294; Email: [email protected]
Nucleic Acids Research, 2001, Vol. 29, No. 23 4809
transducer of checkpoint signals that control cell cycle
progression. However, experiments with mutants in the yeast
Rad53 protein rule against this possibility, at least with respect
to phosphorylation of the middle subunit. Rad53 is a protein
kinase required for the DNA damage and DNA replication
checkpoints in mitotic cells and its activation depends on Mec1
(9,10,33–36). In checkpoint-deficient rad53 mutants, Rfa2
phosphorylation still occurs during the normal cell cycle and in
response to DNA damage or DNA replication inhibition (7),
and Rfa1 phosphorylation still occurs in response to DNA
replication inhibition (8). Therefore, RPA phosphorylation
under these conditions does not correlate with checkpoint
function. Consistent with these results, experiments in human
cells have suggested that IR-induced RPA phosphorylation is
not required for the S phase DNA damage checkpoint (37).
Nonetheless, Rfa1 phosphorylation is compromised in rad53
cells exposed to DNA-damaging agents during G1 (8), a condition under which Rfa1 is known to mediate cell cycle delay
(38). Therefore, certain phosphoisomers of RPA might be
involved in the checkpoint response during specific phases of
the cell cycle.
Given that Mec1 and ATM also function in recombination,
RPA phosphorylation could alternatively be involved in regulating recombination activities. To explore this possibility, we
chose to examine RPA status in yeast cells undergoing
meiosis, a process that includes a high level of programmed
recombination. We now demonstrate that Rfa2 becomes
hyperphosphorylated during meiotic progression through two
distinct reactions. A primary Rfa2 phosphorylation event is
induced early in meiosis that is independent of DNA replication and recombination and does not require Mec1. Subsequently, a secondary Rfa2 phosphorylation reaction is induced
that requires initiation of recombination and is Mec1
dependent. Although Mec1 is necessary for the ‘pachytene
checkpoint’ that halts meiotic progression when recombination
is incomplete (12), we observe extensive Mec1-dependent
Rfa2 phosphorylation in a checkpoint-defective meiotic
recombination mutant. Based on these studies, we suggest that
Mec1-dependent RPA phosphorylation is involved in recombination rather than maintenance of checkpoint delay.
MATERIALS AND METHODS
Yeast strains and growth
The following DSY strains were kindly provided by Drs David
Stuart (University of Alberta) and Curt Wittenberg (The
Scripps Research Institute) (13): DSY1089 (wild-type), MATa/α
ho::LYS2/ ″ lys2/ ″ leu2::hisG/ ″ trp1::hisG/ ″ ura3/ ″ arg4-BglII/
arg4-NspI his4B/his4X; DSY984 (clb5 clb6), MATa/α
ho::LYS2/ ″ lys2/ ″ LEU2/leu2 trp1::hisG/ ″ ura3/ ″ arg4/ ″
clb5::URA3/ ″ clb6::TRP1/ ″; DSY1057 (mec1), MATa/α
ho::LYS2/ ″ lys2/ ″ leu2::hisG/ ″ ura3/ ″ arg4-BglII/arg4-NspI
his4B::LEU2/his4X::LEU2::URA3 mec1-1/ ″ smlx/ ″; DSY1037
(mec1), MATα ho::LYS2 lys2 leu2::hisG ura3 arg4-BglII
his4B::LEU2 mec1-1 smlx. The following DKB strains were
the generous gift of Dr Douglas Bishop (University of Chicago)
(39,40): DKB98 (wild-type), MATa/α ho::LYS2/ ″ lys2/ ″
leu2::hisG/ ″ ura3/ ″ arg4-BglII/arg4-NspI his4B::LEU2/
his4X::LEU2; DKB490 (spo11), MATa/α ho::LYS2/ ″ lys2/ ″ade2/ ″
leu2/ ″ ura3/ ″ his4X-ADE2-his4B/ ″ spo11::hisG-URA3-hisG/ ″;
DKB376 (rad50S), MATa/α ho::LYS2/ ″ lys2/ ″ leu2::hisG/ ″
ura3/ ″ his4B::LEU2/his4X::LEU2-URA3 rad50KI-81::URA3/ ″;
DKB159 (rad50S), MATa ho::LYS2 lys2 leu2::hisG ura3
his4B::LEU2 rad50KI81::URA3; DKB330 (dmc1), MATa/α
ho::LYS2/ ″ lys2/ ″ leu2::hisG/ ″ ura3/ ″ arg4-BglII/arg4-NspI
his4B::LEU2/his4X::LEU2-URA3 dmc1::LEU2/ ″. YGB242
(rad50S mec1) was constructed in our laboratory employing
strains DSY1031 and DKB159 and has the genotype MATa/α
ho::LYS2/ ″ lys2/ ″ leu2::hisG/ ″ ura3/ ″ his4B::LEU2/ ″ mec1-1/ ″
smlx/ ″ rad50KI81::URA3/ ″. YGB138 (wild-type) is a diploid
W303 strain (41): MATa/α ade2-1/ ″ can1-100/ ″ his3-11,115/ ″
leu2-3,112/ ″ trp1-1/ ″ ura3-1/ ″. With the exception of YGB138,
all strains are derived from SK-1 (42).
YPD and YPG media for yeast growth were prepared as
described (43). YPA for presporulation growth consisted of
1% yeast extract, 2% bactopeptone and 2% potassium acetate.
Sporulation medium (SPM) for SK-1 cells included 0.3% (w/v)
potassium acetate, 0.02% (w/v) raffinose, 2.5 × 10–3% (w/v)
leucine, and each of arginine, histidine, tryptophan and uracil
at 5 × 10–4% (w/v). An established sporulation protocol was
employed to allow for synchronous progression of SK-1 cells
through meiosis (13,44). Diploid cells were first streaked from
a frozen stock onto a YPG plate and incubated at 30°C. A
single colony was selected and grown in YPD medium overnight at 30°C and the YPD culture was used to inoculate YPA
medium. The YPA culture was incubated in an air shaker at
30°C, 200 r.p.m. for 12 h from a starting OD600 of ∼0.2 or for
16 h from a starting OD600 of ∼0.1. The cells were harvested by
centrifugation at 1900 g for 5 min at room temperature, washed
with SPM, resuspended in SPM at the approximate density of
the overnight YPA culture and then incubated at 30°C,
200 r.p.m. A similar protocol was employed for the YGB138
time course. Where indicated, cells were resuspended in SPM
with hydroxyurea (HU) (Toronto Research Chemicals) added
to a final concentration of 0.1 M from a 1 M stock; an equal
volume of water was added to controls.
Western blot analysis
Cells (0.5–1.0 ml) were harvested by centrifugation at 14 000 g
for 5 min at 4°C, washed with 1 ml of cold distilled water and
stored at –70°C. To prepare denatured extract, cells were
resuspended with 50 µl of sample buffer for SDS–PAGE and
then subjected to three cycles of freezing (dry ice or liquid
nitrogen for 2 min) followed by heating (∼95°C for 2 min). The
extracts were spun at 14 000 g for 5 min at room temperature and
aliquots of the supernatant were analyzed by western blotting.
Proteins were resolved by SDS–PAGE employing a 12% polyacrylamide gel (150:1 w/w acrylamide:bisacrylamide) and
then transferred to nitrocellulose in either 25 mM Tris,
192 mM glycine, 20% methanol or 10 mM CAPS–NaOH,
pH 11, 10% methanol. Immobilized RPA subunits were
detected by autoradiography after incubation with anti-Rfa1
and anti-Rfa2 polyclonal primary antibodies, horseradish
peroxidase-linked goat anti-rabbit secondary antibody (Pierce
Chemical Co.) and chemiluminescence reagents (Pierce Chemical Co.). Antisera included our previously described anti-Rfa1
and anti-Rfa2 preparations (8) and an anti-Rfa2 antiserum
kindly provided by Dr Steven Brill (Rutgers University). For
the western blot analyses shown in Figures 1–3 and 5, an equal
volume of denatured extract was loaded in each lane. In the
other experiments, we attempted to equalize the detectable
4810 Nucleic Acids Research, 2001, Vol. 29, No. 23
RPA by loading reduced volumes of the 0 h sample or both the
0 and 2 h samples.
Protein phosphatase treatment
Wild-type cells (DSY1089) undergoing synchronous sporulation were harvested at 6 h, resuspended in buffer containing
50 mM Tris–HCl, pH 7.5, 0.5 M NaCl, 2 mM Na4EDTA,
10 mM β-mercaptoethanol, 20% (v/v) glycerol, 0.2 mM PMSF
and vortexed with glass beads at 4°C (10 repeats of 30 s
vortexing followed by 30 s on ice). The resulting suspension
was centrifugally filtered through cheesecloth at 1900 g for
2 min at 4°C and the supernatant of the filtrate was used for
protein phosphatase studies. Reaction mixtures (20 µl)
containing crude extract were assembled on ice and included
50 mM Tris–HCl, pH 7.5, 0.1 mM Na2EDTA, 5 mM DTT,
0.01% Brij 35, and 2 mM MnCl2. Where indicated, 400 U
bacteriophage λ protein phosphatase (λPPase) (New England
Biolabs) and 10 mM sodium orthovanadate were added and the
mixtures were incubated at 30°C for either 0.5 or 2 h. Vanadate
was included for its λPPase inhibitory activity (45) and the
longer incubation time was employed to completely convert
Rfa2 to the unphosphorylated form. Reactions were terminated
by addition of Na4EDTA to 12 mM and RPA in the reaction
mixtures was visualized by western blot analysis.
Analysis of DNA content
Cells were harvested by centrifugation at 14 000 g for 5 min at
4°C, resuspended in 70% ethanol and stored at 4°C. Aliquots of
the fixed cells were washed once with 1 ml of 50 mM Tris–HCl,
pH 7.5, and resuspended in 1 ml of the same buffer. Samples
were then treated with 250 µg RNase A for 1 h at 37°C
followed by 250 µg proteinase K for 1 h at 37°C. The digested
samples were incubated with either 10× SYBR Green I
(Molecular Probes; Figs 1, 4, 6 and 7) or 50 µg/ml propidium
iodide (Figs 2 and 3) at 4°C over 1–3 nights, sonicated briefly
and analyzed by flow cytometry with a Becton Dickinson
FacsCalibur. DNA content histograms were generated using
WinMDI software.
Nuclear staining
Cells that had been fixed with 3.7% formaldehyde (final
concentration) were washed twice with water, resuspended in
water and sonicated briefly. Aliquots were stained with an
equal volume of 100 µg/ml 4,6-diamidino-2-phenylindole
(DAPI) prepared in mounting medium (Sigma Chemical Co.).
The cells were observed by fluorescence microscopy and
nuclear bodies were scored in at least 100 unbudded cells for
each sample. Budded cells were observed in only a subset of
experiments and at a very low percentage of the total cells in
these cases.
RESULTS
RPA becomes phosphorylated during meiosis
Studies employing vegetative yeast cells have indicated that
RPA phosphorylation is catalyzed during normal growth and
also in response to various forms of genotoxic stress (3,7,8).
Because advancement through meiosis includes both a DNA
replication phase and a subsequent recombination phase
initiated by DNA double-strand breaks (44,46), we investi-
gated the possibility that RPA also becomes phosphorylated
during this specialized developmental pathway. When wildtype SK-1 cells are subjected to conditions that result in
synchronous meiotic progression, two Rfa2 isoforms are
generated that have reduced mobility during SDS–PAGE
(Fig. 1A). Note that a lighter exposure of the data presented in
Figure 1A reveals that the majority of Rfa2 at 0 h migrates with
the faster mobility (data not shown, but see Fig. 4A). The
reduced mobility species are abolished upon treatment of
native extract with protein phosphatase (Fig. 1B), indicating
that both forms are Rfa2 phosphoisomers. The two phosphorylated forms of Rfa2 are also detected in wild-type W303 cells
(data not shown), indicating that the pattern of RPA phosphorylation during meiosis is not influenced by strain background. In
addition, we have observed that the general pattern of mitotic
RPA phosphorylation is not altered by differences in strain
background (data not shown).
Although SDS–PAGE detection of phosphorylated species
does not provide an exact measure of stoichiometry, we can
conclude from our data that both a primary phosphorylated and
a secondary hyperphosphorylated species of Rfa2 are generated during meiosis. We compared the timing of Rfa2 phosphorylation and hyperphosphorylation with the timing of
various meiotic events (Fig. 1A, C and D). Primary Rfa2 phosphorylation is initially observed early in meiosis, leading to
modification of ∼50% of the detectable Rfa2 by 3 h (Fig. 1A).
At this time point, very little DNA has been synthesized as
measured by flow cytometric analysis of DNA content
(Fig. 1C). Therefore, the degree of primary Rfa2 phosphorylation does not correlate with the extent of DNA synthesis. In
fact, primary Rfa2 phosphorylation occurs even in the absence
of DNA replication (see below). Further meiotic progression
leads to quantitative Rfa2 phosphorylation and to Rfa2 hyperphosphorylation, which coincides with induction of his4 heteroallelic recombination as measured by detection of histidine
prototrophs in a ‘return-to-growth’ assay (47,48; Fig. 1A and
D). Therefore, the appearance of neither phosphorylated nor
hyperphosphorylated Rfa2 correlates directly with initiation of
DNA synthesis, as has been suggested for Rfa2 phosphorylation during unperturbed vegetative growth (3). Rfa2 dephosphorylation also occurs in a stepwise manner, proceeding from
the hyperphosphorylated to the phosphorylated form when
recombination is complete and from the phosphorylated to the
unphosphorylated form later in meiosis (see 28 h time points in
Figs 2A and 3A).
Primary meiotic RPA phosphorylation is independent of
DNA replication
To gain insight into the role that DNA synthesis plays in
meiotic RPA phosphorylation, we analyzed a strain containing
deletions of the clb5 and clb6 genes. This double mutant is
incapable of replicating DNA during meiosis but continues
through later meiotic events due to a checkpoint defect (13,49).
As shown in Figure 2, clb5 clb6 cells support the early meiotic
Rfa2 phosphorylation reaction with kinetics similar to wildtype despite the defect in meiotic DNA replication. Therefore,
the initial Rfa2 phosphorylation event is independent of DNA
replication. These results further indicate that a cyclindependent kinase activated by either the Clb5 or Clb6 B-type
cyclin does not catalyze this Rfa2 phosphorylation reaction. In
contrast to primary Rfa2 phosphorylation, secondary Rfa2
Nucleic Acids Research, 2001, Vol. 29, No. 23 4811
D
Figure 1. Rfa2 phosphorylation during meiotic progression. (A) Western blot analysis of Rfa2 in synchronously sporulating DSY1089 cells (SK-1 wild-type). In
this time course and all others represented below, 0 h is the time of transfer from YPA to SPM. (B) Phosphatase treatment of native extract from DSY1089 cells
harvested 6 h after transfer to SPM. As indicated, reaction mixtures were incubated for either 0.5 or 2 h. λPPase, bacteriophage λ protein phosphatase. (C) DNA
content analysis by flow cytometry of the DSY1089 culture examined in (A). (D) Recombination and chromosome segregation analysis of the DSY1089 culture
examined in (A). Recombination was measured by scoring histidine prototrophs and progression into meiosis II (MII) was measured by scoring cells with three or
four DAPI staining bodies. cfu, colony forming units.
Mec1 is required for the secondary meiotic RPA
phosphorylation reaction
Figure 2. Primary meiotic Rfa2 phosphorylation in the absence of DNA replication. (A) Western blot analysis of Rfa2 in DSY1089 (wt) and DSY984 (clb5
clb6) cells induced to enter meiosis. P-Rfa2, phosphorylated Rfa2. (B) DNA
content analysis of cultures examined in (A).
phosphorylation is significantly delayed in the clb5 clb6
mutant. It is likely that the absence of normal Rfa2 hyperphosphorylation in these cells results from a recombination
deficiency (50; see below).
Because RPA phosphorylation is dependent on Mec1 during
mitosis (7,8), we analyzed Rfa2 status in a mec1 mutant undergoing meiosis. Although the primary Rfa2 phosphorylation
reaction is unaffected in this mutant, the secondary reaction
leading to Rfa2 hyperphosphorylation is completely abolished
(Fig. 3A). Consistent with previous reports (12,13), the mec1
mutant used in our study proceeds through meiotic S phase
with normal kinetics (Fig. 3B) and is capable of forming
mature asci at later time points (data not shown).
Mutants in mec1 are unable to arrest cell cycle progression
when DNA replication is inhibited during either mitosis or
meiosis (9,13). Therefore, we compared RPA phosphorylation
in wild-type and mec1 cells that had entered meiosis in the
presence of hydroxyurea (HU), an inhibitor of DNA replication. Wild-type cells exhibit substantial primary Rfa2 phosphorylation in the presence of HU, although the extent of this
reaction is compromised relative to that of normal meiotic
progression (Fig. 4A). Interestingly, hyperphosphorylated
Rfa2 is not generated to a significant extent in response to HU,
although we observe a low level of this Rfa2 phosphoisomer at
later time points (Fig. 4A). The pattern of Rfa2 phosphorylation is similar in HU-exposed wild-type and mec1 cells
(Fig. 4C), indicating that Rfa2 phosphorylation in the presence
4812 Nucleic Acids Research, 2001, Vol. 29, No. 23
Figure 3. Mec1 dependence of secondary meiotic Rfa2 phosphorylation.
(A) Western blot analysis of Rfa2 in DSY1089 (wt) and DSY1057 (mec1) cells
induced to enter meiosis. P-Rfa2, phosphorylated Rfa2. (B) DNA content
analysis of cultures examined in (A).
of HU is equivalent to the Mec1-independent primary Rfa2
phosphorylation observed during normal meiotic progression.
Therefore, HU treatment allows for a significant level of
primary but not secondary Rfa2 phosphorylation. Although
Mec1 is required for delay of meiotic progression in response
to HU exposure (13), the paucity of HU-induced Rfa2 hyperphosphorylation suggests that Mec1-dependent Rfa2 phosphorylation does not play an important role in the meiotic DNA
replication checkpoint.
We previously reported that Rfa1 becomes phosphorylated
in response to various forms of genotoxic stress (8). Although
meiotic progression leads to the generation of DNA doublestrand breaks as a prerequisite to recombination (44,46), we
have not detected phosphorylated Rfa1 under these conditions.
However, an Rfa1 phosphoisomer with reduced electrophoretic mobility is detectable at 2 h upon HU exposure, a time
at which untreated cells are beginning to enter S phase in this
experiment (Fig. 4A and B). Similar to Rfa1 phosphorylation
in vegetative cells, this Rfa1 phosphorylation reaction is
dependent on Mec1 (Fig. 4D). Therefore, genotoxic stress
during meiosis, but not the DNA double-strand break ‘damage’
that is naturally induced during meiotic progression, leads to
detectable Mec1-dependent Rfa1 phosphorylation.
Secondary meiotic RPA phosphorylation occurs upon
initiation of recombination
The DNA double-strand breaks required for initiation of
meiotic recombination are catalyzed by Spo11 (51). Interestingly, the absence of recombination initiation in spo11 mutants
is accompanied by a defective pachytene checkpoint (52). Our
analysis of Rfa2 in spo11 cells reveals normal primary Rfa2
phosphorylation but a severe defect in the secondary reaction
(Figs 5 and 6A). Therefore, meiotic Mec1-dependent Rfa2
phosphorylation is dependent on Spo11, most likely due to its
essential function in initiating recombination.
The duplex ends resulting from Spo11-mediated DNA
breakage are processed to generate 3′ single-stranded tails that
can effectively initiate strand exchange. We analyzed meiotic
Figure 4. Meiotic RPA phosphorylation in response to HU exposure.
(A) Western blot analysis of Rfa1 and Rfa2 in DSY1089 cells induced to enter
meiosis in the absence or presence of HU. For this experiment, a single population was resuspended in SPM (0 h) and then divided into untreated (–HU)
and treated (+HU) cultures. P-Rfa1, phosphorylated Rfa1; P-Rfa2, phosphorylated Rfa2. (B) DNA content analysis of cultures examined in (A). Identical 0 h
data are presented in the two histograms. (C) Western blot analysis of Rfa2 in
DSY1089 (wt) and DSY1057 (mec1) cells induced to enter meiosis in the
presence of HU. (D) Western blot analysis of Rfa1 in 6 h samples from the
cultures analyzed in (C). The asterisk indicates an immunoreactive band that is
presumably an Rfa1 degradation product.
Figure 5. Dependence of secondary meiotic Rfa2 phosphorylation on DNA
double-strand break formation. Western blot analysis of Rfa2 in DKB98 (wt)
and DKB490 (spo11) cells induced to enter meiosis. P-Rfa2, phosphorylated
Rfa2.
RPA phosphorylation in rad50S and dmc1 mutants, which are
defective for the resection and strand exchange steps, respectively (39,46,53). As with the spo11 strain, primary Rfa2 phosphorylation appears to be unaffected in either rad50S or dmc1
cells (Fig. 6A). Note that the slight delay in induction of
primary Rfa2 phosphorylation in the mutant strains is likely
explained by their slight delay in meiosis entry as determined
Nucleic Acids Research, 2001, Vol. 29, No. 23 4813
Figure 7. Mec1 dependence of secondary Rfa2 phosphorylation in rad50S
cells. (A) Western blot analysis of Rfa2 in DKB376 (rad50S) and YGB242
(rad50S mec1) cells induced to enter meiosis. P-Rfa2, phosphorylated Rfa2.
(B) DNA content analysis of the cultures examined in (A).
addition, rad50S mutants in the SK-1 background are reported
to progress into meiosis I with either normal or delayed
kinetics, while dmc1 mutants arrest at pachytene in a Mec1dependent manner (12,52–54). The strains used in our study
exhibit the predicted phenotypes, as multinuclear bodies
indicative of chromosome segregation are observed in spo11
and rad50S cells, but not in dmc1 cells (Fig. 6C). Because a
rad50S mec1 mutant generates very little hyperphosphorylated
Rfa2 (Fig. 7), we conclude that Mec1 is required for the vast
majority of secondary Rfa2 phosphorylation in rad50S cells.
Taken together, these studies indicate that the Mec1-dependent
Rfa2 phosphorylation reaction activated upon initiation of
recombination is not sufficient to maintain delay of meiotic
progression.
DISCUSSION
Figure 6. Meiotic Rfa2 phosphorylation in recombination mutants. (A) Western
blot analysis of Rfa2 in DKB98 (wt), DKB490 (spo11), DKB376 (rad50S) and
DKB330 (dmc1) cells induced to enter meiosis. P-Rfa2, phosphorylated Rfa2.
(B) DNA content analysis of the cultures examined in (A). (C) Analysis of
chromosome segregation by DAPI staining of samples from the cultures examined
in (A).
by DNA content analysis (Fig. 6B). We conclude that the
timing of primary Rfa2 phosphorylation is consistent in all
strains employed.
In contrast to the primary reaction, secondary Rfa2 phosphorylation is significantly altered in rad50S and dmc1 cells
relative to the wild-type, as both mutants accumulate a high
level of hyperphosphorylated Rfa2 (Fig. 6A). Although induction
of the secondary reaction appears to be delayed in rad50S and
dmc1 cells, the differences in early meiotic progression
between the mutants and wild-type are likely to account for
this effect (Fig. 6B). Despite the similar Rfa2 hyperphosphorylation in the rad50S and dmc1 strains, rad50S cells
accumulate unresected DNA double-strand breaks while dmc1
cells generate DNA ends with long ssDNA tails (39,46,53). In
During meiosis, DNA is replicated and recombined in reactions that transiently generate ssDNA. Our studies have
revealed that the cellular ssDNA-binding protein RPA
becomes phosphorylated in two successive steps during this
process. These phosphorylation reactions exhibit differences in
both timing and genetic dependencies, indicating that RPA is
modified in a highly controlled fashion during meiosis. Based
on the central role of RPA in DNA replication and recombination and the strictly ordered manner by which these processes
occur during meiosis, it is likely that RPA phosphorylation
plays a regulatory role in meiotic DNA metabolism.
A striking feature of primary Rfa2 phosphorylation is that
nearly all of the detectable Rfa2 becomes modified. The extent
of this reaction is particularly evident in the mec1 mutant,
which is devoid of secondary Rfa2 phosphorylation. The
timing of the primary reaction suggests that an early meiotic
gene encodes an RPA kinase. This hypothesis is supported by
the HU-dependent attenuation of the primary reaction, as a
recent study has shown that early meiotic genes are downregulated by HU treatment (55). Regardless of mechanism, the
complete conversion of Rfa2 to a modified form would suggest
that primary Rfa2 phosphorylation plays a critical role in
4814 Nucleic Acids Research, 2001, Vol. 29, No. 23
meiosis. It is doubtful that the reaction functions in a meiotic
checkpoint process related to DNA metabolism because
primary Rfa2 phosphorylation is not significantly compromised
in the checkpoint-deficient clb5 clb6, mec1, spo11, rad50S or
rad50S mec1 strains. An alternative possibility is that the
primary Rfa2 phosphorylation reaction is required for proper
DNA replication during meiosis. Although the same origins of
DNA replication and many of the same replication proteins are
employed in the mitotic and meiotic S phases, regulation of
these two processes is different and meiotic DNA replication
proceeds at a significantly slower pace than its mitotic counterpart (56,57). DNA replication during mitotic growth serves to
duplicate the genetic material prior to chromosome segregation, while meiotic DNA replication leads to a recombination
phase that precedes two rounds of chromosome segregation.
Studies have shown that meiotic recombination depends on
meiotic DNA replication (50,58) and that the meiotic recombination proteins Spo11 and Rec8 are involved in regulating
meiotic DNA replication (59). Therefore, the sequential processes
of DNA replication and recombination are tightly coupled
during meiosis. Differences in mitotic and meiotic DNA replication, and perhaps RPA phosphorylation, may be related to
differences in the events that directly follow the two S phases,
and it is possible that primary Rfa2 phosphorylation serves as
a molecular switch that converts RPA from a mitotic to a
meiotic DNA replication protein.
There is now strong evidence that yeast RPA plays a role in
transcription as well as DNA metabolism. RPA was purified
from yeast cells as a factor that binds to the URS1 transcriptional repression element (60,61) and has been shown to bind
in vitro to URS sites found upstream of various genes involved
in DNA metabolism (62). A recent report has indicated that a
Mec1-independent, Rad53-dependent Rfa2 phosphorylation
event, which is induced in cells lacking the Set1 chromatin
regulator, decreases RPA–URS interaction and leads to
increased expression of certain DNA repair genes (63). In
addition to its function in transcriptional repression, URS1
serves as an upstream activation sequence of several genes that
are induced early in meiosis (57). Therefore, it will be interesting to determine whether primary RPA phosphorylation,
which occurs early in meiosis and is Mec1 independent,
functions in URS1-mediated transcriptional activation.
In contrast to the primary reaction, secondary Rfa2 phosphorylation is dependent on Mec1 and therefore resembles the Rfa2
phosphorylation reaction that is initially induced during DNA
replication in mitotic cells (3,7). We demonstrate here that the
meiotic Mec1-dependent Rfa2 phosphorylation reaction is
more closely associated with recombination than DNA replication. First, induction of secondary Rfa2 phosphorylation
occurs after the bulk of meiotic DNA replication has been
completed and coincides with induction of recombination.
Secondly, Rfa2 hyperphosphorylation requires Spo11, an
enzyme that catalyzes DNA double-strand break formation
during initiation of meiotic recombination (51). Finally,
mutants that generate unresolved meiotic recombination intermediates accumulate hyperphosphorylated Rfa2 well after
meiotic DNA synthesis is complete. These results indicate that
Mec1-dependent Rfa2 phosphorylation is not likely to
function in meiotic DNA replication. It is also unlikely that
Mec1-dependent Rfa2 phosphorylation functions in the
meiotic DNA replication checkpoint, which requires Mec1
(13), because Rfa2 hyperphosphorylation does not occur to a
significant extent when wild-type cells are treated with HU.
Nonetheless, Mec1-dependent Rfa1 phosphorylation is
observed upon HU exposure and the timing of this reaction
coincides with inhibition of DNA replication. Therefore, it
remains possible that Rfa1 phosphorylation is involved in
regulating meiotic progression through S phase.
Our results are consistent with a model in which DNA structures formed during the process of meiotic recombination lead
to Mec1 activation. Such a mechanism has also been proposed
for the Mec1-dependent phosphorylation of Mek1 (64), a
homolog of Rad53 that is required for meiotic recombination
and the associated pachytene checkpoint (52,65). We previously demonstrated that DNA containing both double- and
single-stranded regions efficiently activates human RPA phosphorylation catalyzed by the DNA-activated protein kinase
catalytic subunit (DNA-PKcs) (66), a structural homolog of
Mec1 (67). In addition, a combination of double-stranded and
single-stranded DNA molecules stimulates ATM-catalyzed
RPA phosphorylation (68,69). Therefore, DNA resembling
recombination intermediates supports RPA phosphorylation
catalyzed by two Mec1-like enzymes from human cells.
Several reports have indicated that Mec1 contains a protein
kinase activity (70–72) and it is possible that Mec1 catalyzes
RPA phosphorylation during meiosis through a mechanism
similar to that of either DNA-PKcs or ATM. RPA readily
forms DNA double-strand break-dependent foci in wild-type,
rad50S and dmc1 cells undergoing meiosis and it is likely that
RPA binds to ssDNA generated at DNA double-strand breaks
(73). Binding of Mec1 to DNA ends, analogous to DNA-PKcs
(74) or ATM (75), would lead to co-localization of Mec1 and
RPA and efficient Rfa2 phosphorylation. In support of such a
model, ATM and RPA have been reported to co-localize on
synapsed meiotic chromosomes in mouse spermatocytes (76).
The dependence of secondary Rfa2 phosphorylation upon
initiation of recombination implies that Mec1-dependent RPA
phosphorylation has a recombination function. Although Mec1
is required for the pachytene checkpoint that delays meiotic
progression when recombination is incomplete (12), our analysis
provides evidence that RPA hyperphosphorylation is not
involved in meiotic delay. Consistent with previous reports
(52–54), we have observed significant chromosomal segregation in the recombination-defective rad50S mutant. The inappropriate meiotic progression of these cells is accompanied by
extensive accumulation of hyperphosphorylated Rfa2, arguing
against a checkpoint function for the secondary Rfa2 phosphoisomer. It is noteworthy that rad50S cells retain a residual
checkpoint activity that delays meiotic progression relative to
wild-type cells (52,54). Both Mec1 and Tel1 are required for
this characteristic meiotic delay (54) and we have shown that
secondary Rfa2 phosphorylation in rad50S cells is also Mec1
dependent. Therefore, we cannot formally exclude the possibility that secondary Rfa2 phosphorylation is involved in the
signal that initiates meiotic delay. Nonetheless, our data clearly
demonstrate that secondary Rfa2 phosphorylation is not
involved in maintaining the pachytene checkpoint.
In the absence of a checkpoint function, an attractive possibility is that Mec1-dependent Rfa2 phosphorylation is directly
involved in regulating the process of meiotic recombination. A
number of studies have suggested that the phosphorylation
state of RPA can affect DNA metabolism, as RPA from human
Nucleic Acids Research, 2001, Vol. 29, No. 23 4815
American Cancer Society and by funds from the Karmanos
Cancer Institute.
REFERENCES
Figure 8. Schematic representation of RPA phosphorylation states. Note that
HU-induced RPA phosphorylation in mitotic cells also involves the Mec1
homolog Tel1 (7,8).
cells exposed to DNA damaging agents is hyperphosphorylated
and does not support efficient SV40 DNA replication in vitro
(6,77,78). Interestingly, our previous studies employing the same
cell-free SV40 system revealed a connection between RPA
phosphorylation and recombination. We demonstrated that DNA
replication-dependent RPA phosphorylation is catalyzed by
DNA-PKcs (66), an enzyme that is required for DNA doublestrand break repair and V(D)J recombination (79–82).
Although our studies suggested that RPA phosphorylation
occurs as a consequence of DNA replication (66), recombination is intimately involved with replication during mitotic S
phase (83,84) and it might be difficult to distinguish a replication
effect from a recombination effect. Therefore, RPA phosphorylation during mitotic S phase could resemble the meiotic reaction by requiring initiation of recombination rather than DNA
replication and could also be involved in regulating recombination. It will be important to determine whether RPA phosphorylation affects any of the functional interactions between RPA
and other meiotic and mitotic recombination proteins (85,86).
A comparison of the predominant forms of phosphorylated
RPA that are observed in untreated and HU-treated wild-type
yeast is depicted in Figure 8. As shown, each condition leads to
the generation of a different RPA phosphoisomer. It is
intriguing that different recombination mechanisms are known
to be favored under different conditions. For example, mitotic
recombination preferentially involves sister chromatids while
meiotic recombination involves homologs, and crossing over
occurs much more frequently in meiosis than mitosis (20,87).
Determining whether RPA phosphorylation is involved in
controlling the relative recombination activities of mitotic
versus meiotic and unperturbed versus stressed yeast cells will
require genetic analysis of RPA phosphorylation site mutants
and biochemical characterization of the various RPA phosphoisomers. These studies will help to define the contribution
of defects in RPA phosphorylation to the mec1 phenotype and,
ultimately, to the AT disease state.
ACKNOWLEDGEMENTS
We thank Drs David Stuart, Curt Wittenberg and Douglas
Bishop for providing yeast strains and Dr Steven Brill for
providing antiserum. We also thank Dr Grant Brown and
Dagmawi Iyasu for critical review of the manuscript. This
work was supported by grant RPG-00-211-01-CCG from the
1. Wold,M.S. (1997) Replication protein A: a heterotrimeric, single-stranded
DNA-binding protein required for eukaryotic DNA metabolism.
Annu. Rev. Biochem., 66, 61–92.
2. Iftode,C., Daniely,Y. and Borowiec,J.A. (1999) Replication protein A
(RPA): the eukaryotic SSB. Crit. Rev. Biochem. Mol. Biol., 34, 141–180.
3. Din,S., Brill,S.J., Fairman,M.P. and Stillman,B. (1990) Cell-cycleregulated phosphorylation of DNA replication factor A from human and
yeast cells. Genes Dev., 4, 968–977.
4. Fotedar,R. and Roberts,J.M. (1992) Cell cycle regulated phosphorylation
of RPA-32 occurs within the replication initiation complex. EMBO J., 11,
2177–2187.
5. Liu,V.F. and Weaver,D.T. (1993) The ionizing radiation-induced
replication protein A phosphorylation response differs between ataxia
telangiectasia and normal human cells. Mol. Cell. Biol., 13, 7222–7231.
6. Carty,M.P., Zernik-Kobak,M., McGrath,S. and Dixon,K. (1994) UV
light-induced DNA synthesis arrest in HeLa cells is associated with
changes in phosphorylation of human single-stranded DNA-binding
protein. EMBO J., 13, 2114–2123.
7. Brush,G.S., Morrow,D.M., Hieter,P. and Kelly,T.J. (1996) The ATM
homologue MEC1 is required for phosphorylation of replication protein A
in yeast. Proc. Natl Acad. Sci. USA, 93, 15075–15080.
8. Brush,G.S. and Kelly,T.J. (2000) Phosphorylation of the replication
protein A large subunit in the Saccharomyces cerevisiae checkpoint
response. Nucleic Acids Res., 28, 3725–3732.
9. Weinert,T.A., Kiser,G.L. and Hartwell,L.H. (1994) Mitotic checkpoint
genes in budding yeast and the dependence of mitosis on DNA replication
and repair. Genes Dev., 8, 652–665.
10. Paulovich,A.G. and Hartwell,L.H. (1995) A checkpoint regulates the rate
of progression through S phase in S. cerevisiae in response to DNA
damage. Cell, 82, 841–847.
11. Siede,W., Allen,J.B., Elledge,S.J. and Friedberg,E.C. (1996) The
Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the
human ATM gene product, is required for G1 arrest following radiation
treatment. J. Bacteriol., 178, 5841–5843.
12. Lydall,D., Nikolsky,Y., Bishop,D.K. and Weinert,T. (1996) A meiotic
recombination checkpoint controlled by mitotic checkpoint genes.
Nature, 383, 840–843.
13. Stuart,D. and Wittenberg,C. (1998) CLB5 and CLB6 are required for
premeiotic DNA replication and activation of the meiotic S/M checkpoint.
Genes Dev., 12, 2698–2710.
14. Kato,R. and Ogawa,H. (1994) An essential gene, ESR1, is required for
mitotic cell growth, DNA repair and meiotic recombination in
Saccharomyces cerevisiae. Nucleic Acids Res., 22, 3104–3112.
15. Bashkirov,V.I., King,J.S., Bashkirova,E.V., Schmuckli-Maurer,J. and
Heyer,W.D. (2000) DNA repair protein Rad55 is a terminal substrate of
the DNA damage checkpoints. Mol. Cell. Biol., 20, 4393–4404.
16. de la Torre-Ruiz,M.A. and Lowndes,N.F. (2000) The Saccharomyces
cerevisiae DNA damage checkpoint is required for efficient repair of
double strand breaks by non-homologous end joining. FEBS Lett., 467,
311–315.
17. Grushcow,J.M., Holzen,T.M., Park,K.J., Weinert,T., Lichten,M. and
Bishop,D.K. (1999) Saccharomyces cerevisiae checkpoint genes MEC1,
RAD17 and RAD24 are required for normal meiotic recombination partner
choice. Genetics, 153, 607–620.
18. Mills,K.D., Sinclair,D.A. and Guarente,L. (1999) MEC1-dependent
redistribution of the Sir3 silencing protein from telomeres to DNA
double-strand breaks. Cell, 97, 609–620.
19. Martin,S.G., Laroche,T., Suka,N., Grunstein,M. and Gasser,S.M. (1999)
Relocalization of telomeric Ku and SIR proteins in response to DNA
strand breaks in yeast. Cell, 97, 621–633.
20. Paques,F. and Haber,J.E. (1999) Multiple pathways of recombination
induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol.
Mol. Biol. Rev., 63, 349–404.
21. Liberi,G., Chiolo,I., Pellicioli,A., Lopes,M., Plevani,P., Muzi-Falconi,M.
and Foiani,M. (2000) Srs2 DNA helicase is involved in checkpoint
response and its regulation requires a functional Mec1-dependent pathway
and Cdk1 activity. EMBO J., 19, 5027–5038.
4816 Nucleic Acids Research, 2001, Vol. 29, No. 23
22. Downs,J.A., Lowndes,N.F. and Jackson,S.P. (2000) A role for
Saccharomyces cerevisiae histone H2A in DNA repair. Nature, 408,
1001–1004.
23. Savitsky,K., Bar-Shira,A., Gilad,S., Rotman,G., Ziv,Y., Vanagaite,L.,
Tagle,D.A., Smith,S., Uziel,T., Sfez,S. et al. (1995) A single ataxia
telangiectasia gene with a product similar to PI-3 kinase. Science, 268,
1749–1753.
24. Gatti,R.A. (1998) Ataxia-telangiectasia. In Vogelstein,B. and
Kinzler,K.W. (eds) The Genetic Basis of Human Cancer.
McGraw-Hill, New York, NY, pp. 275–300.
25. Taylor,A.M.R., Harnden,D.G., Arlett,C.F., Harcourt,S.A.,
Lehmann,A.R., Stevens,S. and Bridges,B.A. (1975) Ataxia telangiectasia:
a human mutation with abnormal radiation sensitivity. Nature, 258,
427–429.
26. Painter,R.B. and Young,B.R. (1980) Radiosensitivity in ataxiatelangiectasia: a new explanation. Proc. Natl Acad. Sci. USA, 77,
7315–7317.
27. Beamish,H. and Lavin,M.F. (1994) Radiosensitivity in ataxiatelangiectasia: anomalies in radiation-induced cell cycle delay. Int. J.
Radiat. Biol., 65, 175–184.
28. Kastan,M.B., Zhan,Q., El-Deiry,W.S., Carrier,F., Jacks,T., Walsh,W.V.,
Plunkett,B.S., Vogelstein,B. and Fornace,A.J.,Jr (1992) A mammalian
cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in
ataxia-telangiectasia. Cell, 71, 587–597.
29. Meyn,M.S. (1993) High spontaneous intrachromosomal recombination
rates in ataxia-telangiectasia. Science, 260, 1327–1330.
30. Luo,C.M., Tang,W., Mekeel,K.L., DeFrank,J.S., Anne,P.R. and
Powell,S.N. (1996) High frequency and error-prone DNA recombination
in ataxia telangiectasia cell lines. J. Biol. Chem., 271, 4497–4503.
31. Morrison,C., Sonoda,E., Takao,N., Shinohara,A., Yamamoto,K. and
Takeda,S. (2000) The controlling role of ATM in homologous
recombinational repair of DNA damage. EMBO J., 19, 463–471.
32. Zhou,B.B.S. and Elledge,S.J. (2000) The DNA damage response: putting
checkpoints in perspective. Nature, 408, 433–439.
33. Zheng,P., Fay,D.S., Burton,J., Xiao,H., Pinkham,J.L. and Stern,D.F.
(1993) SPK1 is an essential S-phase-specific gene of Saccharomyces
cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase.
Mol. Cell. Biol., 13, 5829–5842.
34. Allen,J.B., Zhou,Z., Siede,W., Friedberg,E.C. and Elledge,S.J. (1994)
The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA
damage-induced transcription in yeast. Genes Dev., 8, 2401–2415.
35. Sanchez,Y., Desany,B.A., Jones,W.J., Liu,Q., Wang,B. and Elledge,S.J.
(1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in
yeast cell cycle checkpoint pathways. Science, 271, 357–360.
36. Sun,Z., Fay,D.S., Marini,F., Foiani,M. and Stern,D.F. (1996) Spk1/Rad53
is regulated by Mec1-dependent protein phosphorylation in DNA
replication and damage checkpoint pathways. Genes Dev., 10, 395–406.
37. Morgan,S.E. and Kastan,M.B. (1997) Dissociation of radiation-induced
phosphorylation of replication protein A from the S-phase checkpoint.
Cancer Res., 57, 3386–3389.
38. Longhese,M.P., Neecke,H., Paciotti,V., Lucchini,G. and Plevani,P.
(1996) The 70 kDa subunit of replication protein A is required for the
G1/S and intra-S DNA damage checkpoints in budding yeast.
Nucleic Acids Res., 24, 3533–3537.
39. Bishop,D.K., Park,D., Xu,L. and Kleckner,N. (1992) DMC1:
a meiosis-specific yeast homolog of E. coli recA required for
recombination, synaptonemal complex formation, and cell cycle
progression. Cell, 69, 439–456.
40. Shinohara,A., Gasior,S., Ogawa,T., Kleckner,N. and Bishop,D.K. (1997)
Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have
both distinct and overlapping roles in meiotic recombination. Genes Cells,
2, 615–629.
41. Thomas,B.J. and Rothstein,R. (1989) Elevated recombination rates in
transcriptionally active DNA. Cell, 56, 619–630.
42. Kane,S.M. and Roth,R. (1974) Carbohydrate metabolism during
ascospore development in yeast. J. Bacteriol., 118, 8–14.
43. Adams,A., Gottschling,D.E., Kaiser,C.A. and Stearns,T. (1998)
Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
44. Padmore,R., Cao,L. and Kleckner,N. (1991) Temporal comparison of
recombination and synaptonemal complex formation during meiosis in
S. cerevisiae. Cell, 66, 1239–1256.
45. Zhuo,S., Clemens,J.C., Hakes,D.J., Barford,D. and Dixon,J.E. (1993)
Expression, purification, crystallization, and biochemical characterization
of a recombinant protein phosphatase. J. Biol. Chem., 268, 17754–17761.
46. Cao,L., Alani,E. and Kleckner,N. (1990) A pathway for generation and
processing of double-strand breaks during meiotic recombination in
S. cerevisiae. Cell, 61, 1089–1101.
47. Sherman,F. and Roman,H. (1963) Evidence for two types of allelic
recombination in yeast. Genetics, 48, 255–261.
48. Esposito,R.E. and Esposito,M.S. (1974) Genetic recombination and
commitment to meiosis in Saccharomyces. Proc. Natl Acad. Sci. USA, 71,
3172–3176.
49. Dirick,L., Goetsch,L., Ammerer,G. and Byers,B. (1998) Regulation of
meiotic S phase by Ime2 and a Clb5,6-associated kinase in
Saccharomyces cerevisiae. Science, 281, 1854–1857.
50. Smith,K.N., Penkner,A., Ohta,K., Klein,F. and Nicolas,A. (2001) B-type
cyclins CLB5 and CLB6 control the initiation of recombination and
synaptonemal complex formation in yeast meiosis. Curr. Biol., 11, 88–97.
51. Keeney,S., Giroux,C.N. and Kleckner,N. (1997) Meiosis-specific DNA
double-strand breaks are catalyzed by Spo11, a member of a widely
conserved protein family. Cell, 88, 375–384.
52. Xu,L., Weiner,B.M. and Kleckner,N. (1997) Meiotic cells monitor the
status of the interhomolog recombination complex. Genes Dev., 11,
106–118.
53. Alani,E., Padmore,R. and Kleckner,N. (1990) Analysis of wild-type and
rad50 mutants of yeast suggests an intimate relationship between meiotic
chromosome synapsis and recombination. Cell, 61, 419–436.
54. Usui,T., Ogawa,H. and Petrini,J.H.J. (2001) A DNA damage response
pathway controlled by Tel1 and the Mre11 complex. Mol. Cell, 7,
1255–1266.
55. Lamb,T.M. and Mitchell,A.P. (2001) Coupling of Saccharomyces
cerevisiae early meiotic gene expression to DNA replication depends
upon RPD3 and SIN3. Genetics, 157, 545–556.
56. Collins,I. and Newlon,C.S. (1994) Chromosomal DNA replication
initiates at the same origins in meiosis and mitosis. Mol. Cell. Biol., 14,
3524–3534.
57. Kupiec,M., Byers,B., Esposito,R.E. and Mitchell,A.P. (1997) Meiosis and
sporulation in Saccharomyces cerevisiae. In Pringle,J.R., Broach,J.R. and
Jones,E.W. (eds) The Molecular and Cellular Biology of the Yeast
Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, Vol. 3, pp. 889–1036.
58. Borde,V., Goldman,A.S.H. and Lichten,M. (2000) Direct coupling
between meiotic DNA replication and recombination initiation.
Science, 290, 806–809.
59. Cha,R.S., Weiner,B.M., Keeney,S., Dekker,J. and Kleckner,N. (2000)
Progression of meiotic DNA replication is modulated by
interchromosomal interaction proteins, negatively by Spo11p and
positively by Rec8p. Genes Dev., 14, 493–503.
60. Luche,R.M., Smart,W.C. and Cooper,T.G. (1992) Purification of the
heteromeric protein binding to the URS1 transcriptional repression site in
Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA, 89, 7412–7416.
61. Luche,R.M., Smart,W.C., Marion,T., Tillman,M., Sumrada,R.A. and
Cooper,T.G. (1993) Saccharomyces cerevisiae BUF protein binds to
sequences participating in DNA replication in addition to those mediating
transcriptional repression (URS1) and activation. Mol. Cell. Biol., 13,
5749–5761.
62. Singh,K.K. and Samson,L. (1995) Replication protein A binds to
regulatory elements in yeast DNA repair and DNA metabolism genes.
Proc. Natl Acad. Sci. USA, 92, 4907–4911.
63. Schramke,V., Neecke,H., Brevet,V., Corda,Y., Lucchini,G.,
Longhese,M.P., Gilson,E. and Geli,V. (2001) The set1∆ mutation unveils
a novel signaling pathway relayed by the Rad53-dependent
hyperphosphorylation of replication protein A that leads to transcriptional
activation of repair genes. Genes Dev., 15, 1845–1858.
64. Bailis,J.M. and Roeder,G.S. (2000) Pachytene exit controlled by reversal
of Mek1-dependent phosphorylation. Cell, 101, 211–221.
65. Rockmill,B. and Roeder,G.S. (1991) A meiosis-specific protein kinase
homolog required for chromosome synapsis and recombination.
Genes Dev., 5, 2392–2404.
66. Brush,G.S., Anderson,C.W. and Kelly,T.J. (1994) The DNA-activated
protein kinase is required for the phosphorylation of replication protein A
during simian virus 40 DNA replication. Proc. Natl Acad. Sci. USA, 91,
12520–12524.
67. Hartley,K.O., Gell,D., Smith,G.C., Zhang,H., Divecha,N.,
Connelly,M.A., Admon,A., Lees-Miller,S.P., Anderson,C.W. and
Nucleic Acids Research, 2001, Vol. 29, No. 23 4817
68.
69.
70.
71.
72.
73.
74.
75.
76.
Jackson,S.P. (1995) DNA-dependent protein kinase catalytic subunit: a
relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene
product. Cell, 82, 849–856.
Gately,D.P., Hittle,J.C., Chan,G.K.T. and Yen,T.J. (1998)
Characterization of ATM expression, localization and associated
DNA-dependent protein kinase activity. Mol. Biol. Cell, 9, 2361–2374.
Chan,D.W., Son,S.C., Block,W., Ye,R., Khanna,K.K., Wold,M.S.,
Douglas,P., Goodarzi,A.A., Pelley,J., Taya,Y., Lavin,M.F. and
Lees-Miller,S.P. (2000) Purification and characterization of ATM from
human placenta: a manganese-dependent, wortmannin-sensitive
serine/threonine protein kinase. J. Biol. Chem., 275, 7803–7810.
Mallory,J.C. and Petes,T.D. (2000) Protein kinase activity of Tel1p and
Mec1p, two Saccharomyces cerevisiae proteins related to the human
ATM protein kinase. Proc. Natl Acad. Sci. USA, 97, 13749–13754.
Paciotti,V., Clerici,M., Lucchini,G. and Longhese,M.P. (2000) The
checkpoint protein Ddc2, functionally related to S. pombe Rad26,
interacts with Mec1 and is regulated by Mec1-dependent phosphorylation
in budding yeast. Genes Dev., 14, 2046–2059.
Wakayama,T., Kondo,T., Ando,S., Matsumoto,K. and Sugimoto,K.
(2001) Pie1, a protein interacting with Mec1, controls cell growth and
checkpoint responses in Saccharomyces cerevisiae. Mol. Cell. Biol., 21,
755–764.
Gasior,S.L., Wong,A.K., Kora,Y., Shinohara,A. and Bishop,D.K. (1998)
Rad52 associates with RPA and functions with Rad55 and Rad57 to
assemble meiotic recombination complexes. Genes Dev., 12, 2208–2221.
Yaneva,M., Kowalewski,T. and Lieber,M.R. (1997) Interaction of
DNA-dependent protein kinase with DNA and with Ku: biochemical and
atomic-force microscopy studies. EMBO J., 16, 5098–5112.
Smith,G.C., Cary,R.B., Lakin,N.D., Hann,B.C., Teo,S.H., Chen,D.J. and
Jackson,S.P. (1999) Purification and DNA binding properties of the
ataxia-telangiectasia gene product ATM. Proc. Natl Acad. Sci. USA, 96,
11134–11139.
Plug,A.W., Peters,A.H.F.M., Xu,Y., Keegan,K.S., Hoekstra,M.F.,
Baltimore,D., de Boer,P. and Ashley,T. (1997) ATM and RPA in meiotic
chromosome synapsis and recombination. Nature Genet., 17, 457–461.
77. Park,J.S., Park,S.J., Peng,X., Wang,M., Yu,M.A. and Lee,S.H. (1999)
Involvement of DNA-dependent protein kinase in UV-induced replication
arrest. J. Biol. Chem., 274, 32520–32527.
78. Liu,J.S., Kuo,S.R., McHugh,M.M., Beerman,T.A. and Melendy,T. (2000)
Adozelesin triggers DNA damage response pathways and arrests SV40
DNA replication through replication protein A inactivation. J. Biol.
Chem., 275, 1391–1397.
79. Kirchgessner,C.U., Patil,C.K., Evans,J.W., Cuomo,C.A., Fried,L.M.,
Carter,T., Oettinger,M.A. and Brown,J.M. (1995) DNA-dependent kinase
(p350) as a candidate gene for the murine SCID defect. Science, 267,
1178–1183.
80. Lees-Miller,S.P., Godbout,R., Chan,D.W., Weinfeld,M., Day,R.S.,
Barron,G.M. and Allalunis-Turner,J. (1995) Absence of p350 subunit of
DNA-activated protein kinase from a radiosensitive human cell line.
Science, 267, 1183–1185.
81. Blunt,T., Finnie,N.J., Taccioli,G.E., Smith,G.C.M., Demengeot,J.,
Gottlieb,T.M., Mizuta,R., Varghese,A.J., Alt,F.W., Jeggo,P.A. and
Jackson,S.P. (1995) Defective DNA-dependent protein kinase activity is
linked to V(D)J recombination and DNA repair defects associated with
the murine scid mutation. Cell, 80, 813–823.
82. Peterson,S.R., Kurimasa,A., Oshimura,M., Dynan,W.S., Bradbury,E.M.
and Chen,D.J. (1995) Loss of the catalytic subunit of the DNA-dependent
protein kinase in DNA double-strand-break-repair mutant mammalian
cells. Proc. Natl Acad. Sci. USA, 92, 3171–3174.
83. Marians,K.J. (2000) Replication and recombination intersect. Curr. Opin.
Genet. Dev., 10, 151–156.
84. Rothstein,R., Michel,B. and Gangloff,S. (2000) Replication fork pausing
and recombination or “gimme a break”. Genes Dev., 14, 1–10.
85. Sung,P., Trujillo,K.M. and Van Komen,S. (2000) Recombination factors
of Saccharomyces cerevisiae. Mutat. Res., 451, 257–275.
86. Gasior,S.L., Olivares,H., Ear,U., Hari,D.M., Weichselbaum,R. and
Bishop,D.K. (2001) Assembly of RecA-like recombinases: distinct roles
for mediator proteins in mitosis and meiosis. Proc. Natl Acad. Sci. USA,
98, 8411–8418.
87. Kadyk,L.C. and Hartwell,L.H. (1992) Sister chromatids are preferred over
homologs as substrates for recombinational repair in Saccharomyces
cerevisiae. Genetics, 132, 387–402.