8/9/2019 Finally, Polyubiquitinated PCNA Gets Recognized
http://slidepdf.com/reader/full/finally-polyubiquitinated-pcna-gets-recognized 1/2
Finally, Polyubiquitinated PCNA Gets Recognized
Michelle K. Zeman1 and Karlene A. Cimprich1,*1Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA *Correspondence: [email protected]://dx.doi.org/10.1016/j.molcel.2012.07.024
Studies from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell , together with Weston
et al. (2012), reveal that the translocase ZRANB3/AH2 can recognize K63-linked polyubiquitinated PCNA
and plays an important role in restarting stalled replication forks.
DNA damage presents a challenge to
genome integrity during all cell-cycle
phases, but lesions encountered during
DNA replication can be particularly prob-
lematic. These lesions stall the replica-
tion fork, leading to unstable structuresprone to rearrangement and mutation
( Figure 1 A) ( Branzei and Foiani, 2010 ). In
order to prevent this, cells have evolved
ways of stabilizing the stalled fork and
promoting the resumption of DNA replica-
tion. The DNA damage tolerance (DDT)
pathway is a key contributor to this
process, orchestrating lesion bypass
through posttranslational modification of
the replicative clamp, PCNA. Intriguingly,
ithas beenknown for manyyears thatpol-
yubiquitination of PCNA with a K63-linked
chain signals an error-free form of lesion
bypass via template switching ( Ulrich
and Walden, 2010 ). However, the exact
function of the polyubiquitinated PCNA,
and the mechanism behind this form of
lesion bypass, has long been a mystery.
This month, three papers—from Ciccia
et al. (2012) and Yuan et al. (2012) in this
issue of Molecular Cell , and from Weston
et al. (2012) in Genes and Development —
characterize new biochemical activities
and substrates of ZRANB3/AH2, which
have significant implications for the role
of PCNA polyubiquitination and for the
molecular mechanism behind templateswitching.
Although ZRANB3/AH2 has been previ-
ously described as an annealing helicase
or translocase capable of ‘‘rewinding’’
denatured single-stranded DNA (ssDNA)
in vitro ( Yusufzai and Kadonaga, 2010 ),
little was known about its roles in vivo.
Collectively, the current studies show
that ZRANB3/AH2 is recruited to sites of
DNA damage through an interaction with
PCNA, in order to promote fork restart
after fork stalling. This recruitment is
mediated by three domains. Two are re-
quired for direct interaction with PCNA:
a conserved PCNA-interacting protein
(PIP) box, and a C-terminal AlkB2 PCNA-
interaction motif (APIM). The third is an
NPL4 zinc finger (NZF), a specializedtype of ubiquitin-binding domain which
can specifically recognize K63-linked
ubiquitin chains. This is one of the most
interesting findings, as Ciccia et al. show
that this NZFmotif is required for a specific
interaction with the K63-linked polyubi-
quitinated form of PCNA in vitro and for
retention of ZRANB3 at damage sites
in vivo. They also show this association
has functional consequences, as these
motifsare required forefficientfork restart
in cells.
Both the current and previous work
suggests multiple ways by which
ZRANB3/AH2 might act to promote fork
restart. Its ability to reanneal ssDNA
‘‘bubbles’’ has been speculated to regu-
late the balance between wound and
unwound parental DNA at a stalled fork.
This type of activity could oppose the
replicative helicase and other unwinding
activities to stabilize the fork structure
and minimize the accumulation of ssDNA
( Driscoll and Cimprich, 2009 ). Interest-
ingly, however, Ciccia et al. also report
that ZRANB3/AH2 exhibits translocase
activity on two additional substrates,a finding which could have implications
for fork restart. First, ZRANB3/AH2 can
regress stalled forks, which could facili-
tate lesion bypass by providing access
to the newly replicated sister chromatid.
This would allow the cell to avoid
the damaged DNA entirely by using the
undamaged chromatid as a template ( Fig-
ure 1B). Given the specificity of ZRANB3/
AH2 for binding polyubiquitinated PCNA,
a critical signal for template switching at
stalled forks, it is exciting to postulate
that fork regression may be triggered by
recruitment of ZRANB3/AH2 to this modi-
fication. Second, Ciccia et al. show that
ZRANB3/AH2 can disrupt D-loop struc-
tures in vitro. This raises the possibility
that ZRANB3/AH2 prevents unnecessaryrecombination events by dissolving inap-
propriate D loops at the stalled replica-
tion fork or possibly at gaps left behind
the fork ( Figure 1C). Consistent with this
idea, ZRANB3/AH2 is shown to suppress
sister-chromatid exchanges, common
crossover events during perturbed repli-
cation ( Ciccia et al., 2012 ). While sister-
chromatid exchanges are not deleterious
to the cell per se, a higher rate of D-loop
formation increases the likelihood of
inaccurate strand invasion and, by exten-
sion, the chance for alteration of genetic
information.
Surprisingly, the paper from Weston
et al. (2012) also reveals a novel function
for ZRANB3/AH2 as a structure-specific
endonuclease. The ability of ZRANB3/
AH2 to cut replication fork structures
in vitro relies on its HNH motif, a function-
ally divergent domain found in a variety
of DNA-binding proteins. The authors
suggest that this endonuclease activity,
in conjunction with fork regression, may
contribute to the removal of DNA lesions
( Figure 1D). As such a model involves
the repair of DNA damage at the fork,rather than lesion bypass, this finding
could suggest that PCNA polyubiquitina-
tion plays a role in replication-associated
DNA repair as well as DDT.
ZRANB3/AH2 is the second annealing
helicase to be characterized, following
SMARCAL1/HARP ( Driscoll and Cim-
prich, 2009 ), and the work of Yuan et al.
(2012) suggests that there are at least
two more members of this family,
Rad54L and SMARCA1. All four proteins
contain a HARP-like (HPL) domain, which
Molecular Cell 47 , August 10, 2012 ª2012 Elsevier Inc. 333
Molecular Cell
Previews
8/9/2019 Finally, Polyubiquitinated PCNA Gets Recognized
http://slidepdf.com/reader/full/finally-polyubiquitinated-pcna-gets-recognized 2/2
was originally found to confer
annealing helicase activity
to SMARCAL1/HARP ( Ghosal
et al., 2011 ). In addition,
translocases from other fami-lies, such as HLTF and
FANCM, are also capable of
regressing forks ( Unk et al.,
2010 ). This raises the ques-
tions: Why does the cell
need such a variety of seem-
ingly redundant players?
Could these translocases be
working in a damage- or
sequence-specific manner?
In different DNA compart-
ments? With different molec-
ular partners? At least in the
case of SMARCAL1/HARPand ZRANB3/AH2, it seems
clear that these are not
redundant proteins. The
mechanisms and kinetics of
recruitment to stalled forks
are distinct, and they are
not functionally epistatic in
several assays ( Ciccia et al.,
2012, Yuan et al., 2012 ).
Finally, although ZRANB3/
AH2 exhibits a specific pref-
erence for K63-polyubiquiti-
nated PCNA, it is not clear whether
ZRANB3/AH2 is functioning as a new
component in the DDT pathway, in a
parallel DNA repair pathway, or with
something else entirely. HLTF, a key ubiq-
uitin ligase and translocase in the DDT
pathway, also has fork regression activity,
is important for fork restart, and can poly-
ubiquitinate PCNA ( Unk et al., 2010 ).
What, then, is the role of ZRANB3/AH2?
This may be clarified through DNA muta-
tion analysis and epistasis studies with
other DDT proteins. Electron microscopy
and physical interaction studies may
also help reveal if ZRANB3/AH2 can
actively promote fork regression in vivo,
or if its biochemical pro-
perties are modulated differ-
ently in cells. Clearly, how-
ever, these studies open
many new avenues of inves-tigation by linking PCNA
polyubiquitination to specific
biochemical activities and
by beginning to address the
long-standing question of
what recognizes polyubiquiti-
nated PCNA.
REFERENCES
Branzei, D., and Foiani, M. (2010).Nat. Rev. Mol. Cell Biol. 11,208–219.
Ciccia, A., Nimonkar, A.V., Hu, Y.,Hajdu, I., Achar, Y.J., Izhar, L.,Petit, S.A., Adamson, B., Yoon,J.C., Kowalczykowski, S.C., et al.(2012). Mol. Cell 47 , this issue,396–409.
Driscoll, R., and Cimprich, K.A.(2009). Genes Dev. 23, 2359–2365.
Ghosal, G., Yuan, J., and Chen, J.(2011). EMBO Rep. 12, 574–580.
Ulrich, H.D., and Walden, H. (2010).Nat. Rev. Mol. Cell Biol. 11,479–489.
Unk, I., Hajdu , I., Blastya k, A., and Haracska, L.
(2010). DNA Repair (Amst.) 9, 257–267.
Weston, R.,Peeters, H., and Ahel, D. (2012). GenesDev. 15, 1558–1572.
Yuan, J., Ghosal, G., and Chen, J. (2012). Mol. Cell 47 , this issue, 410–421.
Yusufzai,T., and Kadonaga,J.T. (2010). Proc. Natl. Acad. Sci. USA 107 , 20970–20973.
C Recombination-Mediated
Template SwitchingSister chromatid exchange
A Stalled Fork
B Regressed ForkLesion bypass
Fork restart Z
D Repaired ForkLesion repair
Fork restart
Regressed Fork
Nicked Fork
A n n e a l i n g
a
c t i v i t y N u c l e
a s e
a c t i v i t y
D i s r u p t i o
n o f
D - l o
o p s
Figure 1. Fork Restart Activities of ZRANB3/AH2(A) In the presence of DNA damage (red star), the replication fork stalls, allow-
ing for the accumulation of RPA (brown) on single-stranded DNA. PCNA
(purple) is polyubiquitinated by proteins in the DNA damage tolerance
pathway to induce template switching. According to the current work, polyu-
biquitinated PCNAis recognized by ZRANB3/AH2 (green),which mayremodel
the fork and promote fork restart in several ways.
(B) ZRANB3/AH2 demonstrates fork regression activity in vitro, which may
facilitate template switching and lesion bypass in cells.
(C) ZRANB3/AH2 is capable of disrupting D-loop structures in vitro, an activity
which may prevent sister chromatid exchanges in vivo.
(D) ZRANB3/AH2 exhibits structure-specific endonuclease activity on the
leading strand of replication fork structures in vitro (green triangle), which, in
conjunction with fork regression, may lead to repair of the lesion at the stalled
replication fork.
334 Molecular Cell 47 , August 10, 2012 ª2012 Elsevier Inc.
Molecular Cell
Previews
