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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:   cimprich@stanford.eduhttp://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

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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.

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