©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
and the activation and co-ordination of the various cellular events
that comprise the DNA Damage Response (DDR). These include
the reorganization of chromatin and the formation of IR induced
[Cell Cycle 7:21, 3399-3404; 1 November 2008]; ©2008 Landes Bioscience
The ubiquitination of PCNA is an essential event in the
operation of the DNA Damage Tolerance (DDT) pathway that
is activated after DNA damage caused by UV or chemical agents
during S-phase. This pathway allows the bypass of DNA damage
by translesion synthesis that would otherwise cause replication
fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and
polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway.
Mono-and polyubiquitination of PCNA are key processes in the
translesion bypass and template switching sub-pathways of the
DDT. DNA damage by IR causes DSBs, which trigger the DNA
Damage Response (DDR). The ubiquitin ligase RNF8 has a critical
role in the assembly of BRCA1 complexes at the DSBs in the DDR.
We show that RNF8 readily mono-ubiquitinates PCNA in the
presence of UbcH5c, and polyubiquitinates PCNA in the added
presence of Ubc13/Uev1a. These reactions are the same as those
performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion
suppressed both UV and MNNG-stimulated mono-ubiquitination
of PCNA, revealing that an RNF8-dependent pathway for PCNA
ubiquitination is operative in vivo. These findings provide evidence
that RNF8, a key E3 ligase in the DDR, may also play a role in
Living organisms are constantly subjected to DNA damage from
external agents and internal processes, and complex mechanisms
have evolved for the maintenance of genomic integrity. DSBs are
caused by ionizing radiation, oxygen reactive species as well as
chemical agents. Their formation triggers the assembly of multi-
protein complexes that are involved in the sensing of DNA damage,
foci, the activation of cell cycle checkpoints,1-3 the recruitment of
DNA repair machinery, as well as the regulation of mechanisms that
dictate apoptosis.4-6 The initial events are the sensing of DSBs by
the MRN complex and the recruitment and activation of the ATM
(ataxia telangiectasia mutated) checkpoint kinase. This leads to phos-
phorylation of histone H2AX and the recruitment of MDC1.1 This
first stage is followed by recruitment of p53BP1 and the BRCA1 A
complex, BRCA1/BARD1/CCDC98 (Abraxias)/RAP80/BRCC36,
to IR induced foci and initiation of checkpoint responses and DNA
repair.5-7 Recent studies have provided a model for the recruitment
of the BRCA1 complex in which RNF8 plays a key role as a regu-
lator that links the two assembly stages.7-10 RNF8 (RING finger
protein 8) is a member of the RING domain group of E3 ubiquitin
ligases that also possesses a FHA (forkhead associated) domain.11-13
The model that has emerged is that RNF8 is recruited to the DNA
damage complex via binding of its FHA domain to phosphothre-
onines on MDC1, and underscores the widening roles of protein
ubiquitination in the cellular responses to DNA damage.2 RNF8, in
combination with Ubc13, then polyubiquitinates histones 2A and
H2AX. This event allows the recruitment of the BRCA1 A complex
via RAP80, which possesses two ubiquitin interacting motifs that
recognize K63 polyubiquitin chains.
DNA damage that results in modification of DNA in S-phase cells
is particularly deleterious to cell survival. The encounter of the repli-
cation forks with template lesions produced by genotoxic agents such
as UV damage and alkylating agents causes replication fork stalling,
that in turn can lead to further DNA damage and cell death.14,15
Replication fork stalling triggers a specific S-phase checkpoint by
activation of the ATR (ataxia telangiectasia and Rad3 related) check-
point kinase that leads to slowing of the S-phase and inhibition of
DNA synthesis.3 The DNA damage tolerance (DDT) pathway, in
which ubiquitination of PCNA plays a central role, is evoked to
avoid prolonged stalling of replication forks at base lesions.16 PCNA
functions as a molecular sliding clamp for DNA polymerases δ and
ε at the replication fork, and is involved in numerous other DNA
transactions.17 The DDT pathway allows the bypass of base lesions
in a process that requires the mono-ubiquitination of PCNA by the
E3-E2 pair Rad18-Rad6, which then leads to recruitment of the
translesion polymerases.16,18,19 A second, less well understood, DDT
sub-pathway requires the polyubiquitination of PCNA and involves
*Correspondence to: Marietta Y.W.T. Lee; Department of Biochemistry and Molecular
Biology; New York Medical College; Valhalla, New York 10595 USA; Email:
Submitted: 08/29/08; Accepted: 09/08/08
Previously published online as a Cell Cycle E-publication:
PCNA is ubiquitinated by RNF8
Sufang Zhang, Jennifer Chea, Xiao Meng, Yajing Zhou, Ernest Y.C. Lee and Marietta Y.W.T. Lee*
Department of Biochemistry and Molecular Biology; New York Medical College; Valhalla, New York USA
Abbreviations: PCNA, proliferating cell nuclear antigen; RNF8, RING finger protein 8; DDR, DNA damage response; DDT, DNA damage
tolerance pathway; DSB, double stranded break; MRN, Mre11-Rad50-Nbs1, BRCA1, breast cancer 1; BARD1, BRCA1 associated RING
domain 1; MDC1, mediator of DNA damage checkpoint 1; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine
Key words: PCNA, RNF8, ubiquitination, DNA damage response, DNA damage tolerance, translesion bypass, cell cycle checkpoint
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Uev1a to function as the E2 enzyme in place of UbcH5c for the
mono-ubiquitination of PCNA were examined. None of these func-
tioned as an E2 partner with RNF8 for the mono-ubiquitination of
PCNA (Fig. 2D). Ubc13 is a known partner of RNF8,13 and normally
functions as a heterodimer with a Uev (ubiquitin E2 variant) partner,
for which we used Uev1a, a homolog of yeast Mms2.16 The inability
of Ubc13 to mono-ubiquitinate PCNA (despite its ability to interact
with RNF8) is consistent with evidence that it only acts on mono-
ubiquitinated substrates, e.g., in the Rad5Ubc13 polyubiquitination
of PCNA22 and in the auto polyubiquitination of BRCA1.26
RNF8 ubiquitinates PCNA
Polyubiquitination of PCNA by RNF8 and Ubc13/Uev1a. The
polyubiquitination of PCNA via K63 linked chains in the DDT
pathway is catalyzed by Rad5 and a Ubc13 heterodimer with either
Mms2 or Uev1a. This requires the prior mono-ubiquitination of
PCNA by Rad18-Rad6.16 We therefore tested the ability of Ubc13/
Uev1a to polyubiquitinate PCNA in the presence of RNF8 and
UbcH5c. The time course of formation of mono-ubiquitinated
PCNA by RNF8-UbcH5c alone is shown in Figure 3A (left). When
Ubc13/Uev1a was also present, PCNA was polyubiquitinated
(Fig. 3A, right). The appearance of polyubiquitinated PCNA was
delayed by comparison with the formation of mono-ubiquitinated
PCNA, consistent with a stepwise reaction. These results show that
polyubiquitination of PCNA by RNF8-Ubc13/Uev1a requires prior
mono-ubiquitination of PCNA, i.e., mono-ubiquitinated PCNA is
the substrate for polyubiquitination. PCNA is mono-ubiquitinated
at K164 by RNF8-UbcH5c and polyubiquitinated by RNF8-Ubc13/
Uev1a via K63 isopeptide linkages. We tested the PCNA-K164R
mutant as a substrate for RNF8-UbcH5c and found that it is neither
mono- (Fig. 3B, lanes 2 and 4) nor polyubiquitinated (Fig. 3B,
lane 6). This shows that ubiquitination of PCNA takes place by
modification of K164, similar to the ubiquitination of PCNA by
Rad18-Rad6.16 Next, we demonstrated that PCNA was polyubiquit-
inated via K63 isopeptide linked ubiquitin chains by RNF8-Ub13/
Uev1a. This was determined by the use of the K48R, K63R, K48R/
K63R and K0 (all lysines mutated to R) ubiquitin mutants. The
use of these mutants would not be expected to affect the mono-
ubiquitination of PCNA, as shown for ubi-K63R (Fig. 3C, lane 3).
Polyubiquitination of PCNA was supported by ubi-K48R (Fig. 3C,
template switching for error free bypass of DNA lesions.15,16 PCNA
is polyubiquitinated by Rad5-Ubc13/Mms2 in yeast,16 and by the
human Rad5 homologs, SHPRH20 or HLTF21 and human Ubc13,
which forms a heterodimer with Mms2 or Uev1a.22
We examined the possibility that RNF8, which plays a major role
in the assembly of the DDR protein complexes at DSBs, also plays
a role in PCNA ubiquitination. We show that RNF8, in concert
with UbcH5c, catalyzes the mono-ubiquitination of PCNA, and in
concert with Ubc13/Uev1a, polyubiquitinates PCNA. Depletion of
RNF8 in A549 cells led to suppression of PCNA mono-ubiquitina-
tion after UV or MNNG treatment, providing evidence for a novel
RNF8-dependent pathway for PCNA ubiquitination.
PCNA is mono-ubiquitinated by RNF8 and UbcH5c. The
hypothesis that RNF8 might function as an E3 ubiquitin ligase that
acts on PCNA was tested in a reconstituted in vitro system using
purified recombinant human proteins (Experimental Procedures).
The assay contained E1 (ubiquitin activating enzyme), RNF8 as the
E3, and UbcH5c as the E2 ubiquitin conjugating enzyme. UbcH5c
was selected as it catalyzes the auto-ubiquitination of RNF8.11 In
addition, it is extensively used for in vitro assays as it interacts with
many E3 ligases23,24 PCNA was efficiently ubiquitinated by RNF8-
UbcH5c in a reconstituted assay system (Experimental Procedures),
as shown by the time course of the reaction (Fig. 1A). The sole
product was mono-ubiquitinated PCNA, and the reaction was near-
complete by 1 h; no evidence for the presence of polyubiquitinated
PCNA was found even after 2 h. The identity of the mono-ubiq-
uitinated form of PCNA was confirmed by Western blotting with
antibody against ubiquitin (Fig. 1B).
The dependence of the reaction on RNF8 concentration was
examined. The reaction was near-complete at 150 ng RNF8/assay
(Fig. 2A). The dependence of the reaction on PCNA concentra-
tion was examined over a range of 5 to 120 ng/assay (Fig. 2B). The
formation of product was maximal by ca. 80 ng/15 μl assay, which is
equivalent to 23 nM PCNA monomer. This concentration of PCNA
is comparable to that which we routinely used for the assay of DNA
polymerase δ on the poly(dA)oligo(dT) template/primer (100 μg/30
μl assay); in this system the apparent Kd for PCNA was ca. 17 nM.25
An important conclusion that can be drawn is that RNF8 interacts
with PCNA with sufficient affinity to allow the robust reaction that
we observed. RNF8, like other RING E3 ligases, functions as a scaf-
fold and does not possess a catalytic site, so that it must engage in
a protein-protein interaction with PCNA in the reconstituted assay.
The ability of RNF8 to bind PCNA was confirmed by overlay blot-
ting with digoxigenin-PCNA (Fig. 2C).
The abilities of UbcH2, UbcH3 and UbcH6, RAD6 or Ubc13/
3400 Cell Cycle 2008; Vol. 7 Issue 21
Figure 1. Mono-ubiquitination of PCNA by RNF8. (A) Mono-ubiquitination of
PCNA by RNF8 and UbcH5c. The ubiquitination of PCNA was assayed in a
reconstituted system using purified human proteins (Experimental Procedures).
The reaction mixtures contained 10 μg of ubiquitin, 30 nM E1 ubiquitin acti-
vating enzyme, 500 nM of UbcH5c, 100 ng of PCNA and 100 ng of RNF8.
After incubation at 30°C for the indicated times, the reaction products were
examined by SDS-PAGE and Western blotting with anti-PCNA (Experimental
Procedures). Positions of PCNA and mono-ubiquitinated PCNA are shown
by the arrowheads. (B) Confirmation of the identity of mono-ubiquitinated
PCNA. The blot shown in (A) was stripped and re-blotted with anti-ubiquitin.
(The high molecular weight products are polyubiquitinated products that are
expected from the auto-polyubiquitination of RNF8).
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
lane 5), but not by ubiK63R/K48R or ubi-K0 (Fig. 3C, lanes 6 and
7). This demonstrates that the polyubiquitination of PCNA occurs
via K63 isopeptide linkages.
Depletion of RNF8 results in suppression of DNA damaged
induced PCNA mono-ubiquitination. The preceding results estab-
lish that RNF8 functions as an E3 ligase that is able to mono-and
polyubiquitinate PCNA, thereby revealing an alternate biochemical
route for the established reactions catalyzed by Rad18-Rad6 and
Rad5-Ubc13 in the DDT pathway.16 In order to determine whether
RNF8 plays an in vivo role in the ubiquitination of PCNA after
RNF8 ubiquitinates PCNA
www.landesbioscience.comCell Cycle 3401
DNA damage, we examined the mono-ubiquitination of PCNA in
A549 cells in which RNF8 was depleted. shRNAs against RNF8
were used to isolate clonal populations of cells in which RNF8 was
depleted (Experimental Procedures). Western blot analysis for RNF8
depletion is shown for seven of these cell lines in Figure 4A. Next,
the cells were treated with UV to elicit PCNA monoubiquitination.
PCNA ubiquitination was suppressed to apparent near-completion
after UV treatment (Fig. 4B, lanes 3, 4, 7 and 8; C, lanes 6–8) as
compared to control A549 cells, or with cells that were transfected
with control shRNA. The effects of the alkylating agent, MNNG,
were also examined in three cell lines in which RNF8 was depleted.
A similar suppression of PCNA mono-ubiquitination was observed
(Fig. 4C, lanes 2–4). These results reveal the presence of an RNF8-
dependent pathway for PCNA mono-ubiquitination in vivo.
The near-complete suppression of UV-induced PCNA mono-
ubiquitination by RNF8 depletion is similar in extent to that which
has been previously reported in cells in which Rad18 is ablated. This
was demonstrated in Rad18-/- mouse fibroblast cell lines27 and in
HeLa and 293T cells in which Rad18 was depleted by siRNA.28
This presents an incongruity if both RNF8 and Rad18 are simply
involved in the ubiquitination of PCNA. However, it is possible that
RNF8 (or Rad18) may also play a role upstream of the ubiquitina-
tion of PCNA, e.g., in the assembly of the signaling/repair complexes
at stalled replication fork structures in analogy with those formed at
DSBs in the DDR. (It may be noted that only a small fraction of
PCNA is typically observed to be ubiquitinated after UV damage,
as seen in Figure 4B, and the presence of active deubiquitinating
enzymes can also affect the levels of ubiquitination products that
are recovered. Thus, caution should be exercised in regard to the
question of the completeness of the suppression of PCNA mono-
ubiquitination by either RNF8 or Rad18 depletion.) Evidence for
a redundancy in the Rad18 pathway for PCNA ubiquitination has
been demonstrated in avian DT40 cells. In DT40 Rad18 null cells,
PCNA ubiquitination was reduced, but not completely suppressed
in response to MMS (methyl methane sulfonate) treatment of DT40
Rad18 null cells.29 In the latter study USP1, the enzyme that is
responsible for deubiquitination of PCNA,30 was also genetically
deleted. When the resulting Rad18/usp1 DT40 cell line was treated
with MMS, PCNA ubiquitination was only partially reduced,
allowing a clear demonstration for a RAD18 independent pathway
for PCNA ubiquitination.29
Our studies show that the E3-E2 pair RNF8-UbcH5 mono-ubiq-
uitinates PCNA, which is then polyubiquitinated by RNF8-Ubc13/
Uev1a. These findings are significant, as these reactions recapitulate
those catalyzed by Rad18-Rad6 and Rad5-Ubc13/Mms2 in the
well-established system involved in the DDT pathway.16 Worthy
of comment is that RNF8-UbcH5c is only able to catalyze mono-
ubiquitination of PCNA, whereas UbcH5c commonly performs
polyubiquitination reactions with other ubiquitin ligases, e.g., in
BRCA1/BARD1 autoubiquitination.31 Structural analysis of the
UbcH5c-ubiquitin complex has revealed that it has a second ubiq-
uitin binding site that is required for the assembly of polyubiquitin
chains.24,31 (In the case of Ubc13, this second site is provided
by the Uev partner).32 Disruption of the non-catalytic ubiquitin
binding site of UbcH5c by site-directed mutagenesis blocked
Figure 2. Dependence of the Mono-Ubiquitination Reaction on PCNA and
RNF8 Concentrations. (A) RNF8 concentration dependence. The reactions
were performed as in (A) with increasing concentrations of RNF8 from 1 to
300 ng/assay for 1 hr. “C” refers to a control reaction in which ATP was
omitted in this and subsequent panels. (B) PCNA concentration dependence.
The reactions were performed as in (A) with increasing concentrations of
PCNA from 5 to 120 ng/assay for 1 hr. “C” refers to the control reaction in
which ATP was omitted. (C) RNF8 interacts with PCNA. The ability of PCNA
to interact with RNF8 was examined by overlay blotting with digoxigenin-
PCNA as previously described.40 Lane M, protein molecular weight markers;
lane 1, glutathione-S-transferase (0.5 μg); lane 2, his-tagged RNF8 (0.5
μg). The specificity of the interaction is supported by the failure to observe
signals for the molecular weight markers and for glutathione-S-transferase.
(D) Rad6, Ubc13/Uev1a, UbcH6, UbcH2 and UbcH3 do not function as
E2 enzymes with RNF8 for the mono-ubiquitination of PCNA. PCNA was
incubated with RNF8 and either UbcH5c (lane1), Rad6 (lane2), Ubc13/
Uev1a (lane 3), UbcH6 (lane 4), UbcH2 (lane5) or UbcH3 (lane 6), as
described in Experimental Procedures. Products were analyzed by Western
blotting with anti-PCNA.
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
mono-ubiquitination after DNA damage treatments with UV or
MNNG. Our findings are significant, as RNF8 had been relatively
unstudied.11-13 Its cellular functions are now beginning to emerge
with the discovery of its role in the DDR that initiates the G2/M
checkpoint in response to DSBs.4-6 RNF8 is one of only two human
E3 ligases that possess both FHA and RING domains, the other being
the mitotic checkpoint protein Chfr (checkpoint with forkhead and
ring finger domains). Chfr is also involved in G2/M arrest when cells
are subjected to microtubule stress by nocadazole, but not in response
to DSBs.34 In this regard, RNF8 has also been shown to play a role in
RNF8 ubiquitinates PCNA
3402Cell Cycle 2008; Vol. 7 Issue 21
the regulation of mitotic exit.35 Our discovery that RNF8 is involved
in PCNA ubiquitination now extends its potential functions into the
DDT pathway. The latter is an S-phase checkpoint response that is
activated by DNA damage caused by UV and other genotoxic agents.
Unlike the DDR, less is known of the assembly of the complexes that
regulate the S-phase checkpoint. Thus, the involvement of RNF8,
a component of the IR induced DDR, in the DDT pathway is
consistent with a potential sharing of components between the two
pathways that may represent points of integration of the pathways. In
this regard, it is relevant that RNF8 has been shown to be involved
in UV-induced p53BP1 targeting and phosphorylation, and in the
activation of the UV dependent G2/M checkpoint.36
In summary, these studies have established the biochemical capa-
bility of RNF8 to ubiquitinate PCNA in a reconstituted system,
self polyubiquitination of BRCA1, and only mono-ubiquitinated
BRCA1 was formed.31 Analysis of the mono- or polyubiquitina-
tion of BRCA1 by UbcH5c and several other E2 enzymes have
provided further insights into the structural basis for why a given
E3-E2 complex would catalyze mono- or polyubiquitination. E2
enzymes which lack the non-catalytic ubiquitin binding site only
catalyzed mono-ubiquitination of BRCA1, unlike UbcH5c which
is able to catalyze both mono-and polyubiquitination.26 By analogy,
the restriction of the RNF8-UbcH5c reaction to monoubiquitina-
tion could be explained by spatial interference of PCNA with the
UbcH5c non-catalytic ubiquitin binding site. This explanation could
also involve spatial interference with UbcH5c carrying pre-assembled
If RNF8 exerts a role in PCNA ubiquitination in vivo via its
ability to mono-ubiquitinate PCNA, the identity of the E2 ligase
with which it interacts remains to be determined. UbcH5c is an
attractive candidate for the E2 that could be involved in mono-
ubiquitination of PCNA, as we have eliminated several candidates,
and because mono-ubiquitination places further restrictions on the
E3-E2 pair.26 Ubc13 itself requires a Uev partner, and it has two
possible partners, Mms2 and Uev1a in human cells. It has been
reported that Mms2 is involved in DNA damage repair, while Uev1a
is involved in NFkappaB activation.22 Another unknown Uev may
also be involved, as depletion of both Mms2 and Uev1a did not
abrogate PCNA polyubiquitination.28 Similarly, checkpoint protein
assembly by RNF8/Ubc13 has been reported to be independent of
Mms2 and Uev1a.33
Our data support a role for RNF8 in PCNA ubiquitination in
vivo, since shRNA depletion of RNF8 was shown to block PCNA
Figure 3. Polyubiquitination of PCNA by RNF8. (A) PCNA is polyubiquit-
inated by RNF8-Ubc13/Uev1a after its prior monoubiquitination by RNF8-
UbcH5c. PCNA was incubated with RNF8 and UbcH5c, or with RNF8,
UbcH5c and Ubc13/Uev1a for periods of 1, 2 and 3 h and analyzed for
formation of ubiquitinated PCNA. “C” refers to a control reaction in which
ATP was omitted. Positions of the protein molecular weight markers (kDa)
are shown on the right for all panels. (B) PCNA is ubiquitinated on K164.
The ubiquitination of PCNA and its K164R mutant by RNF8 were assayed
as described in Experimental Procedures. His-PCNA and his-PCNA-K164R
were used for this experiment. Ubiquitination by RNF8-UbcH5c: Lane 1, his-
PCNA with ATP omitted; lanes 2 and 4 (duplicates), his-PCNA-K164R; lane
3, his-PCNA. Ubiquitination by RNF8, UbcH5c and Ubc13/Uev1a: lane
5, his-PCNA; lane 6, his-PCNA-K164R. Ubiquitination by RNF8-Ubc13/
Uev1a: lane 7, his-PCNA; lane 8, his-PCNA-K164R. (C) Polyubiquitination
of PCNA occurs through K63 isopeptide linkages. PCNA was ubiquitinated
using ubiquitin and its K64R, K48R, K48R/K64R or K0 mutants. Assays were
performed with RNF8 and UbcH5c (lanes 1–3) or with RNF8, UbcH5c and
Ubc13/Uev1a (lanes 4–7). [The greater amount of polyubiquitinated PCNA
observed with the ubi-K48R mutant (lane 5) compared to ubiquitin (lane 4) is
likely due to the fact that the RNF8 auto-ubiquitination reaction is suppressed,
i.e., competition for the ubiquitin substrate is reduced].
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Reconstituted system for PCNA ubiquitination by RNF8. The
reaction mixtures (15 μl) contained 10 μg of ubiquitin, 30 nM E1
ubiquitin activating enzyme, 500 nM of UbcH5c as the E2 ubiquitin
conjugating enzyme unless otherwise stated, 100 ng RNF8, 100 ng
of PCNA or his-PCNA, 20 ng of ubiquitin aldehyde, 1 mM ATP,
5 mM MgCl2, 40 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol.
Where used as the E2 enzymes, Rad6, UbcH2, UbcH6 or UbcH3
were also added at 500 nM concentrations. Assays were performed
at 30°C for 1 h. The proteins were then resolved by SDS-PAGE on
10% polyacrylamide gels, followed by Western blot analysis with
RNF8 ubiquitinates PCNA
www.landesbioscience.comCell Cycle 3403
PC10 monoclonal anti-PCNA (Santa Cruz Biotechnology). Proteins
were visualized by chemiluminescence (Amersham Biosciences,
Cell culture and shRNA knock down of RNF8. A549 cells
(American Type Culture Collection) were maintained in RPMI
medium 1640 supplemented with 10% fetal bovine serum.39 HuSH
29mer shRNA constructs against human RNF8 and control shRNA
(non-effective shGFP) were designed and synthesized by OriGene
Technologies, Inc., (Rockville, MD). The RNF8-shRNA target
sequences were as follows:
The selection of A549 cells in which RNF8 was knocked down
was performed according to the protocol supplied by OriGene
Technologies. A549 cells were plated on 6 cm dishes and transfected
with the control and RNF8 shRNA plasmids in duplicate using
Lipofectamine 2000 (Invitrogen, Carlsbard, CA). The medium was
replaced after 24 hours with fresh growth medium containing 2 μg/
ml puromycin, and selection pressure maintained for 2–3 weeks to
obtain single clonal populations. Twelve clonal populations of cells
against RNF8 shRNA or control shRNA were taken from each plate
for further culture and analysis. The knockdown efficiency was evalu-
ated by Western blotting with rabbit polyclonal antibody against
anti-RNF8 (Abcam Inc., Cambridge, MA).
This work was supported by NIH grants GM31973 and
and have established that PCNA ubiquitination in vivo exhibits a
dependency on RNF8. These findings support the idea that RNF8
may play a role in the DDT pathway for DNA repair.
Proteins. Ubiquitin (Ubi), Ubi-K48R, Ubi-K0, Ubi-K63R/
K48R, Ubc13/Uev1a, UbcH2, UbcH3 and UbcH6 were purchased
from Boston Biochem. (Cambridge, MA). Recombinant human
PCNA was expressed in Escherichia coli and purified as described
previously.25,37 His6-PCNA was expressed by insertion of the
PCNA coding sequence into the pET28b vector (Novagen, EMD
Chemicals, Gibbstown, NJ) between the NdeI and EcoRI sites.
His6-PCNA was expressed in E. coli BL21star. The his6-PCNA-
K164R mutant was generated by the QuikChange site directed
mutagenesis kit (Stratagene,Delaware, DE). The coding sequences of
UbcH5c and Rad6 were cloned into pET28b and expressed in E. coli
as his-tagged proteins. His-tagged RNF8 and E1 ubiquitin activating
enzyme (Uba1) were expressed in Sf9 cells using the pFastBac HT
vectors in the Bac-to-Bac system according to the manufacturer’s
protocols (Invitrogen, Carlsbad, CA). Sf9 cells were grown as previ-
ously described.25,38 His-tagged proteins were purified on Ni-NTA
beads (Qiagen Inc, Valencia, CA). RNF8 was further purified by
FPLC chromatography on a Mono-Q anion exchange column. All
the proteins were purified to near-homogeneity.
Figure 4. RNF8 is required for the Mono-ubiquitination of PCNA that is Induced by DNA Damage Treatments with UV or MNNG. (A) Suppression of RNF8
expression by RNAi interference. A549 cells were transfected with vectors for four 29 mer shRNAs (Experimental Procedures). RNF8 levels were examined
by Western blotting with anti-RNF8 for the seven clonal cell populations that were used in this experiment. These were R3-12, R4-9, R4-10, R2-6, R4-2, R4-3
and R4-4, where the prefixes R2, R3 and R4 refer to the shRNA used, and the suffixes refer to the isolates. “RC” refers to A549 cells that were transfected
with control shRNA. (B) RNF8 knockdown suppresses mono-ubiquitination of PCNA after UV treatment. R3-12, R4-9, R4-10 and R2-6 cells were treated with
UV (20 J/m2) and harvested for analysis four hours later and Western blotted for PCNA. “RC”, cells transfected with control shRNA. “C”, parent A549 cells
that were not transfected. (C) RNF8 knockdown suppresses mono-ubiquitination of PCNA after MNNG treatment. The R4-2, R4-3 and R4-4 cell lines were
treated with 100 ng/ml MNNG for 4 h, and Western blotted for PCNA (lanes 2–4). A parallel experiment was performed where the cells were UV-treated
as in (B) (lanes 6–8). The positions of PCNA and mono-ubiquitinated PCNA are indicated by arrowheads.
©2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
mono-or specific polyubiquitin chain linkages. Nat Struct Mol Biol 2007; 14:941-8.
27. Watanabe K, Tateishi S, Kawasuji M, Tsurimoto T, Inoue H, Yamaizumi M. Rad18 guides
poleta to replication stalling sites through physical interaction and PCNA monoubiquitina-
tion. EMBO J 2004; 23:3886-96.
28. Brun J, Chiu R, Lockhart K, Xiao W, Wouters BG, Gray DA. hMMS2 serves a redundant
role in human PCNA polyubiquitination. BMC Mol Biol 2008; 9:24.
29. Simpson LJ, Ross AL, Szuts D, Alviani CA, Oestergaard VH, Patel KJ, Sale JE. RAD18-
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