Fixin' to Divide
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Mohammad, Duaa H., and Michael B. Yaffe. “Fixin’ to Divide.”
Molecular Cell 45, no. 3 (February 2012): 273–275. © 2012
Elsevier Inc.
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http://dx.doi.org/10.1016/j.molcel.2012.01.022
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Final published version
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Fri May 27 02:09:29 EDT 2016
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http://hdl.handle.net/1721.1/91632
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Fixin’ to Divide
Duaa H. Mohammad1 and Michael B. Yaffe1,*
1David H. Koch Institute for Integrative Cancer Research, Departments of Biology and Biological Engineering, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA
*Correspondence: myaffe@mit.edu
DOI 10.1016/j.molcel.2012.01.022
In this issue of Molecular Cell, Yata et al. (2012) show that the mitotic kinase and cell-cycle regulator Plk1 can
directly stimulate the DNA repair process, providing a potential mechanism of crosstalk between DNA repair
and cell-cycle signaling.
The mitotic kinase Plk1 acts as a central
choreographer for every substage of
mitosis as well as subsequent events
during cytokinesis. In addition, Plk1 is required for DNA-damaged cells to re-enter
mitosis after repair is concluded and cellcycle checkpoint signaling is silenced.
What no one would have expected, however, is a paradoxical role for this mitotic
kinase in stimulating the repair process
itself.
DNA damage activates checkpoint
pathways—ATM-Chk2, ATR-Chk1, and
p38MAPK-MK2—that disable cyclin/
Cdks, resulting in cell-cycle arrest in G1,
S, or G2. In addition, Plk1 activity is inhibited by the DNA damage response.
After DNA repair, Plk1 drives mitotic entry
by inhibiting the activity of these checkpoint kinase pathways (van Vugt and
Medema, 2005). Simply put, the G2
checkpoint pathways directly oppose
cyclin/CDK activity, and the checkpoint
kinases that do this are shut down by
the activity of Plk1. DNA repair is also
modulated by the cell-cycle response.
Repair differs depending on the cell-cycle
stage; double-strand DNA breaks (DSBs)
in G1, for example, are repaired by
error-prone nonhomologous end-joining
(NHEJ). In contrast, during S and G2, repair by error-free homologous recombination (HR) can occur because of the
availability of the sister chromatid (Moynahan and Jasin, 2010). During HR, 50 to 30
resection of the DSB results in activation
of the ATR arm of the DNA damage
response as well as the generation of an
HR-competent DNA template. For HR to
occur in an S- and G2-dependent manner,
the cell-cycle machinery and the DNA
damage response must intersect. Mre11
and CtIP are two such molecules that
‘‘read’’ both cell-cycle state and DNA
damage signaling. Mre11, an exo- and
endonuclease component of the Mre11/
Rad50/Nbs1 (MRN) complex that is involved in both damage signaling and
repair, specifically associates with cyclin
A/Cdk2 during late S and G2, the HR
competent points in the cell cycle. Similarly, CtIP, which associates with DSBs
exclusively at S and G2, is phosphorylated
by Cdk2 to generate a BRCA1 phosphodocking site and stabilize CtIP itself (Buis
et al., 2012; Sartori et al., 2007). The resulting Mre11/CtIP/BRCA1 complex resects
the break, creating a DNA molecule with
30 single-stranded overhangs competent
for RPA loading. BRCA2 then loads
Rad51 onto the RPA-coated molecule
(Figure 1) and inhibits Rad51’s ATPase
activity to stabilize the Rad51-ssDNA
complex. This complex, in turn, invades
the sister chromatid, ultimately repairing
the damage through homologous
recombination (Moynahan and Jasin,
2010; Shin et al., 2003). In this issue,
a fascinating new paper by Yata and
colleagues now implicates Plk1 phosphorylation of Rad51 in the same process
(Yata et al., 2012).
The findings of Yata et al. are
surprising, given that the major accepted
role of Plk1 is driving cells from G2 into M
phase, a stage of the cell cycle where
DNA damage signals thought to be
required for recruitment of repair
machinery are suppressed. Furthermore,
Plk1 is known to phosphorylate 53BP1,
a molecule important for NHEJ, blocking
its ability to form foci after DNA damage
(van Vugt et al., 2010). Using a series of
in vitro kinase assays, Yata et al. now
demonstrate that Rad51 is also
a substrate of Plk1. Plk1 phosphorylation
of Rad51 at S14 primes Rad51 for subsequent CK2 phosphorylation at T13. This
CK2-phosphorylated form of Rad51 can
then bind directly to the FHA- and tandem
Molecular Cell 45, February 10, 2012 ª2012 Elsevier Inc. 273
Molecular Cell
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DSB
sister chromatid
cyclin A/Cdk2, Mre11, CtIP
5’-3’ resection
RPA loading
+
BRCA2
Rad51
CK2
Plk1
Rad51
pSer-14
pThr-13
pSer-14
Possible
release of
Rad51
monomers
NBS1
BRCA2
Figure 1. A Model for DSB Repair by HR Using BRCA2-Dependent and Plk1/CK2-Dependent Pathways
DSBs generated in S and G2 undergo 50 / 30 resection by the activity of cyclin A/Cdk2, Mre11, and CtIP. The 30 single-strand overhang is then coated with RPA.
RPA is displaced by Rad51 through BRCA2- or Plk1/Ck2-dependent events involving destabilization of Rad51 oligomers, loading of Rad51 monomers onto the
ssDNA, and inhibition of Rad51 ATPase activity. Plk1 and CK2 phosphorylation of Rad51 enhances Rad51 recruitment to sites of DNA damage through a putative
interaction with NBS1.
BRCT domain-containing N terminus of
Nbs1. These findings led the authors to
infer that Rad51 phosphorylation by CK2
might recruit both Rad51 and Nbs1
proteins to foci in a manner similar to
that previously reported for CK2-dependent interactions between Nbs1 and
MDC1 (Lloyd et al., 2009; Williams et al.,
2009). In agreement with this, phosphorylation of endogenous Rad51 at the Plk1
and CK2 sites could be observed in cells
following nocodazole arrest, and briefly
following 4 Gy IR. Evidence for a direct
Rad51-Nbs1 interaction in vivo, however,
still awaits conformation, something that
could be difficult given the relatively high
Kd of the complex and its potentially transient nature.
In agreement with the model proposed
by Yata et al., Rad51 mutagenesis studies
confirmed roles for the Plk1 phosphorylation site in both Rad51 foci formation and
in DNA repair. Cells expressing nonphosphorylatable T13A and S14A mutants of
Rad51 had reduced Rad51 foci formation;
conversely, the phosphomimetic Rad51
S14D mutant displayed increased foci
formation. Intriguingly, the S14A Rad51
mutant fared the worst in a clonogenic
survival assay after 4 Gy IR, even in the
background of WT Rad51, while the
S14D mutants had enhanced survival.
Together, these data indicate that phosphorylation of Rad51 at an in vitro Plk1
site enhances Rad51 foci formation and
survival after IR and that nonphosphor-
274 Molecular Cell 45, February 10, 2012 ª2012 Elsevier Inc.
ylatable S14A mutants of Rad51 have
dominant-negative effects.
The authors further postulated that
Plk1 and CK2 phosphorylation may result
in BRCA2-independent recruitment of
Rad51 to chromatin (Figure 1). In the
background of WT endogenous Rad51,
BRCA2 knockdown resulted in a reduction
of all forms of Rad51 foci formation, with
the S14A variant most dependent on
BRCA2 expression and the S14D mutant
least dependent. BRCA2-deficient cells
are known to be exquisitely sensitive to
PARP inhibitors, and resistance to PARP
inhibitors is shown to develop through
the reactivation of HR pathways. Yata
and colleagues therefore examined the
effects of PARP inhibitors on BRCA2
Molecular Cell
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knockdown cells expressing the various
Rad51 mutants. The S14D phosphomimetic mutant of Rad51 led to better
survival than WT Rad51, while the S14A
and T13A mutant cells survived worse,
implying that Plk1 and CK2 phosphorylation of Rad51 could be a mechanism by
which BRCA2-deficient cells acquire resistance to PARP inhibitors. Intriguingly,
in a recent issue of Molecular Cell,
D’Andrea and colleagues described a
novel antirecombinase protein, PARI,
that stimulates Rad51 ATPase activity to
unload Rad51 from ssDNA (Moldovan
et al., 2012). Perhaps Plk1- and CK2dependent phosphorylation of Rad51
interferes with PARI binding, stabilizing
the Rad51-ssDNA filaments independently of BRCA2.
On the basis of these observations, one
would expect phosphorylated forms of
Rad51 to promote HR. Paradoxically,
however, using two different HR reporter
assays, Yata and colleagues found that
all the different mutations in Rad51—
both phospho-mimicking and nonphosphorylatable analogs—showed reduced
HR relative to cells expressing WT
Rad51. The authors interpret this as
evidence that dynamic phosphorylation
and dephosphorylation of Rad51 is likely
important for its HR-promoting function.
Exactly when Plk1 and Rad51 would function in concert to facilitate HR is unclear.
Perhaps Plk1 phosphorylation of Rad51
marks specific times in the cell cycle
when HR activity can be enhanced—
when damage occurs very late in G2 prior
to Plk1 inactivation by checkpoint
kinases, for example, or upon Plk1 reactivation and silencing of the checkpoint
during the late stages of recovery from
damage. Alternatively, Plk3, a Plk1 paralog known to be specifically activated
by DNA damage (Xie et al., 2001), may
be the relevant S14 kinase under some
conditions. In either case, it seems clear
that the potential roles for Plk1 as a master
regulator of the cell cycle after DNA
damage have expanded to include
disruption of 53BP1 foci formation,
enhancement of Rad51 foci formation,
inactivation of Chk1 and Chk2, and reactivation of cyclin/CDK activity. Yata
et al.’s findings of a Plk1-Rad51 connection show that the roles of Plk1 include
more than just modulating the machinery
of mitosis itself.
Lloyd, J., Chapman, J.R., Clapperton, J.A., Haire,
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Buis, J., Stoneham, T., Spehalsky, E., and
Ferguson, D.O. (2012). Nat. Struct. Mol. Biol.,
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Peroxiredoxins as Molecular Triage Agents,
Sacrificing Themselves to Enhance
Cell Survival During a Peroxide Attack
P. Andrew Karplus1,* and Leslie B. Poole2,*
1Department
of Biochemistry and Biophysics, Oregon State University, 2011 Ag Life Sciences Building, Corvallis, OR 97331, USA
of Biochemistry, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA
*Correspondence: karplusp@ucs.orst.edu (P.A.K.), lbpoole@wakehealth.edu (L.B.P.)
DOI 10.1016/j.molcel.2012.01.012
2Department
In this issue of Molecular Cell, Day et al. (2012) reveal a surprising benefit of peroxiredoxin inactivation at high
H2O2, showing that in Schizosaccharomyces pombe turning off peroxide defenses preserves the pool of
reduced thioredoxin for repairing proteins vital to survival.
Hydrogen peroxide (H2O2) is most generally known as a bacteriocide for cleaning
cuts—though it stings like blazes—but it
is also widely promoted for its ‘‘miraculous’’ curative properties. This Yin and
Yang of hydrogen peroxide is also seen in
eukaryotic cell biology, as it causes oxidative damage and cell death at high concentrations, but is also synthesized
Molecular Cell 45, February 10, 2012 ª2012 Elsevier Inc. 275