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Contents lists available at ScienceDirect
DNA Repair
j ournal homepage: www.elsevier.com/locate/dnarepair
Analysis of SHPRH functions in DNA repair and immunoglobulin diversification
a a b b,1
Nils-Sebastian Tomi , Kathrin Davari , David Grotzky , Friedemann Loos ,
a b a,∗
Katrin Böttcher , Samantha Frankenberger , Berit Jungnickel
a
Department of Cell Biology, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich-Schiller University Jena,
Hans-Knoell-Strasse 2, 07745 Jena, Germany
b
Institute of Clinical and Molecular Biology, Helmholtz Center Munich, Marchioninistrasse 25, 81377 Munich, Germany
a
r t i c l e i n f o a b s t r a c t
Article history: During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of
Received 10 April 2014
proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases
Received in revised form 29 August 2014
for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via
Accepted 23 September 2014
K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH
Available online xxx
in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions
have been reported. We have previously shown that the Rad6 pathway is involved in somatic hyper-
Keywords:
mutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting
SHPRH
PCNA expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do
Ubiquitin not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during
DNA repair DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs
DT40 initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor
treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function.
SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy
of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and
genetic plasticity.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction Histone Linker PHD RING helicase (SHPRH) and/or helicase like
transcription factor (HLTF) in vertebrates [4–6]. Then, error-free
Genome maintenance is based on several complementary DNA bypass of the damage is triggered by a barely understood pathway
repair pathways as well as DNA damage signaling. In combina- that involves recruitment of the ZRANB3 translocase for fork restart
tion, these processes ensure that DNA is largely damage-free before [1,7].
being replicated. If a replication fork does encounter DNA damage, While Rad18 plays a substantial role in PCNA monoubiquiti-
its bypass is coordinated by the Rad6 pathway [1]. Monoubiq- nation [8], the existence of at least one alternative E3 ligase for
uitination of the sliding clamp proliferating cell nuclear antigen this process has been postulated [9], and two candidates have
(PCNA) facilitates efficient binding of translesion polymerases har- been proposed [10,11]. Importantly, PCNA monoubiquitination
boring a flexible catalytic site, allowing direct bypass even of bulky may enhance the efficacy of other repair pathways such as the
lesions [2,3]. Depending on the type of lesion and the polymerase Fanconi anemia pathway [12] and non-conservative mismatch
used, this pathway may be rather error-prone. Alternatively, PCNA repair, the latter also occurring outside of S phase [13,14]. So far,
may be polyubiquitinated via K63-linkage in ubiquitin, employing no differential contribution of the different E3 ligases to these
the Ubc13/Mms2 dimer and the E3 ligase Rad5 in yeast, or SNF2 different effects of PCNA monoubiquitination has been reported.
Interestingly, though, Rad18 also has other functions in the reg-
ulation of DNA double strand break repair [15–17] and e.g. viral
∗ infection [18]. For the E3 ligases SHPRH and HLTF, involved in
Corresponding author. Tel.: +49 3641 949 960; fax: +49 3641 949 962.
PCNA polyubiquitination, different reports have also pointed at
E-mail address: [email protected] (B. Jungnickel).
1 redundancy and cooperation as well as other functions [4,5,19,20].
Present address: Department of Reproduction and Development, Erasmus MC,
University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. These ligases belong to the RING family, share a unique domain
http://dx.doi.org/10.1016/j.dnarep.2014.09.010
1568-7864/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
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architecture with Rad5 [5,21,22], and may promote PCNA polyubiq- exon 22 and in addition deleted exons 23–26 of the SHPRH gene,
uitination in in vitro assays or when overexpressed in mammalian aiming at direct inactivation of the crucial RING domain of this E3
cells [5,6,23,24]. While one study reported that both E3 ligases ligase.
−
need to cooperate for efficient PCNA polyubiquitination [5], another All three strategies were attempted in the DT40 V cell line
report detected PCNA diubiquitination even in the absence of lacking the pseudogenes required for Ig gene conversion, which
both enzymes, hinting at a third responsible E3 ligase for PCNA allows measurement of both DNA damage survival and somatic
polyubiquitination [19]. In addition, HLTF was found to enhance hypermutation. While gene inactivation was successful using
PCNA monoubiquitination and recruitment of Polymerase while strategies PHD and RING (Suppl. Tables 1 and 2), targeting effi-
−
inhibiting SHPRH functions upon UV treatment. On the other hand, ciencies for strategy TSS in DT40 V were too low to obtain
SHPRH was shown to increase polymerase recruitment upon reliable complete knockout clones for subsequent analyses (Suppl.
HLTF degradation after MMS treatment [4]. Table 3). For this knockout strategy, we therefore used DT40Cre1
Clearly, the redundancy of E3 ligases claimed to have a function cells still containing the pseudogenes required for Ig gene conver-
in PCNA polyubiquitination complicates mechanistic studies on sion, as this cell line shows somewhat higher targeting efficiencies
their impact on DNA repair. To exacerbate this issue, E3 ligases are for unknown reasons (Suppl. Table 4 and data not shown).
rather large enzymes containing several functional modules that Southern blot analyses confirmed complete removal of the
are encoded by multiple exons. While in RNAi studies, off-target respective parts of the SHPRH gene (Fig. 1E–G). RT-PCR also con-
effects may never be ruled out completely, knockout approaches firmed lack of transcription of the targeted exons (Fig. 1H–J).
may thus lead to alternative splicing events and production of However, for strategy TSS and PHD, residual transcription
partially functional or dominant negative proteins. Potential DNA downstream of the targeted exons was detected, which may lead to
damage specific functions or effects restricted to certain cell types production of at least partially functional truncated proteins. qRT-
or organs make it even more complicated to study E3 ligases PCR analyses revealed a substantial decrease of transcription in case
involved in PCNA ubiquitination. of the promoter knockout in strategy TSS (Suppl. Fig. 1).
We and others have previously shown that Rad18 and PCNA We therefore directly assessed effects of the knockout strategies
ubiquitination are involved in diversification of immunoglobulin on SHPRH protein expression by western blot analyses. Reactivity
genes by somatic hypermutation [25–28]. In the present study, of the antibody used was demonstrated by its ability to detect an
we have asked whether PCNA polyubiquitination contributes to HA-tagged chicken SHPRH protein expressed in DT40 cells (Suppl.
or counteracts Ig diversification. To this end, we have inactivated Fig. 2), however crossreactivity of the antibody with other proteins
SHPRH by 3 different strategies in chicken DT40 B cells that do may obscure some residual smaller SHPRH fragments, in partic-
not harbor a HLTF ortholog. We show that in this system, SHPRH ular as endogenous SHPRH expression in DT40 appears to be low
may have some effect on spontaneous PCNA ubiquitination in the (Fig. 2A). In case of strategy TSS, we detected disappearance of the
absence of exogenous DNA damage, but does not change the levels full length protein in the knockout clones (Fig. 2A) yet no appear-
of DNA damage-induced PCNA mono- or polyubiquitination. We ance of truncated forms. The same was true for strategy PHD
show that SHPRH plays no role in survival of MMS- or cisplatin- (Fig. 2B). In case of strategy RING (Fig. 2C), in addition to disap-
induced damage processed by base or nucleotide excision repair, pearance of the full length form a truncated protein was detected
but is required for survival of topoisomerase II inhibitors trigger- in the knockout cells at the expected molecular weight for SHPRH
ing DNA double strand breaks. Deletion of only the RING domain lacking a RING domain.
of SHPRH is not sufficient to cause this phenotype. Consistent with Accordingly, while knockout of the promoter of SHPRH in strat-
differential roles in processing of single strand lesions or double egy TSS bears the risk of residual downstream transcription,
strand breaks, SHPRH does not affect somatic hypermutation of Ig potentially leading to low level expression of functional protein(s)
genes, but may have some effect on mismatch tolerance during that might contain most important domains of SHPRH, introduc-
immunoglobulin gene conversion. Our findings support the notion tion of a premature stop codon in strategy PHD and downstream
of at least one additional E3 ligase involved in PCNA polyubiquitina- transcription may lead to residual SHPRH fragments with partial
tion, and highlight the complexity of studying the functions of these functions. In case of strategy RING, a protein lacking the RING
enzymes in different processes linked to genome maintenance. domain but encompassing other functional domains of SHPRH is
definitively produced, and a partial positive or dominant negative
function of this truncated SHPRH can not be excluded. We therefore
2. Results decided to use all three knockout sets for functional analyses.
2.1. Generation of SHPRH knockout cells by 3 different strategies 2.2. Effects of SHPRH inactivation on PCNA ubiquitination
SHPRH is a RING type E3 ligase encoded by a gene with 29 exons, SHPRH has been identified as an E3 ligase involved in PCNA
spanning 47 kb. While no functional domain could be identified by polyubiquitination, which can be detected in ubiquitination reac-
homology search within the region encoded by exons 1–5, exons tions using purified proteins or cells overexpressing SHPRH
6–13 code for SNF2 and PHD domains, while exons 24 and 25 code [5,6,23,24]. However, reproducible detection of PCNA polyubiqui-
for the RING domain and exons 26–29 for a HELICc domain (Fig. 1A). tination in normal cells has proven exceedingly difficult [4]. It was
In order to make sure that our SHPRH knockout impairs protein recently reported, though, that treatment of cells with H2O2 leads
function(s), we decided to use three different strategies. In the strat- to rapid and proficient PCNA monoubiquitination [14]. Using this
egy henceforth called TSS (transcriptional start site) (Fig. 1A and approach, we could also reproducibly detect substantial di- and
B), we removed a region starting 1000 bp upstream of the trans- triubiquitination of PCNA in DT40 cells (Suppl. Fig. 3).
criptional start site and ending at the end of exon 4, aiming at Applying this approach to the knockout sets generated before
inactivation of SHPRH transcription. In the strategy called PHD revealed two different issues. In undamaged cells, knockout via
(Fig. 1A and C), we inserted a premature stop codon into exon 6, at strategies TSS and PHD may potentially lead to some increase
the start of the sequence coding for the first SNF2 domain, and in in basal PCNA monoubiquitination (Fig. 3A and B). This was not
addition deleted the subsequent exons 7, 8 and 9, aiming at inac- observed for strategy RING, a first warning that that the truncated
tivation of all functional domains of the protein. In the strategy protein produced here could have retained some residual function
RING (Fig. 1A and D), we introduced a premature stop codon into (Fig. 3C and data mentioned below). In cells treated with H2O2,
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
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Fig. 1. Inactivation of SHPRH in DT40 cells. (A) Protein and gene structure of SHPRH and indication of the deleted region for each gene targeting strategy. The stop codons
introduced in frame for strategy PHD and RING are displayed. Exons distribution is to scale. (B–D) Gene targeting strategy for deletion of the promoter region (B), the PHD
domain (C) and the RING domain (D). The wild type locus is depicted in the upper drawing and the lower one indicates the targeted integration of the resistance cassette of
the targeting vector shown in the middle. (E) Southern blot of DT40Cre1 SHPRH wild type, two heterozygous and three knockout clones, respectively, using BclI and the probe
−
shown in (B). The wild type allele generates a 5.6 kb fragment and the mutated allele a 3.9 kb fragment. (F) Southern blot of DT40 V SHPRH wild type, two heterozygous
and seven knockout clones, respectively, using BclI and the probe shown in (C). The probe hybridizes to a 5.6 kb and 6.7 kb genomic fragment derived from the wild type and
−
mutated alleles, respectivley. (G) Southern blot analysis of DT40 V SHPRH wild type cells, two heterozygous and three knockout clones, using PvuII and the probe shown
in (D). The wild type allele generates a 6.4 kb fragment and the mutated alleles have a size of 7.4 kb and 10.6 kb, depending on the resistance cassette used. (H–J) Loss of the
targeted exons in the mRNA of the different clones analyzed in (E–G) was confirmed by RT-PCRs. Primer pairs outside of the targeted region indicate residual transcription
of the untargeted region.
SNF2: sucrose non fermentable; H15: histone 1 and 5; PHD: plant homeodomain; RING: really interesting new gene; HELICc: helicase superfamily c-terminal domain; UTR:
untranslated region, arrows indicate primer binding sites.
we observed some variability in between clones of one knock- 2.3. Effects of SHPRH inactivation on DNA repair
out set (e.g. Fig. 3C) as well as in different experiments for the
same cells (Suppl. Fig. 4). Overall, the sensitivity of the assay sys- To assess the function of SHPRH in DNA damage tolerance, we
tem only allows to conclude that the function of SHPRH in DNA performed colony survival assays in methylcellulose medium. We
damage-induced PCNA polyubiquitination can clearly be replaced could not confirm the recently reported function of SHPRH in MMS
by another E3 ligase even in cells lacking HLTF. survival [6] in any of our 3 knockout sets, even though an effect
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
Repair (2014), http://dx.doi.org/10.1016/j.dnarep.2014.09.010
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−/−
Fig. 2. Loss of SHPRH protein expression in SHPRH cells. Western blot analyses of SHPRH expression in the clones derived from the knockout strategies TSS (A), PHD
(B) and RING (C). For strategy TSS, comparison of endogenous expression levels with those obtained by the expression vector are shown. For strategy PHD, the clones
are shown as two sets according to the two heterozygous clones from which they were generated. The estimated band of SHPRH (192 kDa) is marked with an arrow and the
asterisk indicates an unspecific band. A truncated SHPRH protein (∼159 kDa) appears in strategy RING.
−/− K164R
of inactivation of the Rad6 pathway, using Rad18 or PCNA result in etoposide sensitivity (Fig. 4I), once again indicating that
mutants, was clearly seen (Fig. 4A–C). We additionally used cis- this truncated protein retains at least partial functionality. Inter-
platin treatment, as this drug shows a stronger effect in mutants in estingly, overexpression of SHPRH in wild type (Fig. 2A) as well as
the Rad6 pathway [29], but once again no effect of SHPRH inac- TSS knockout cells also interferes with etoposide-, but not MMS-
tivation was detectable (Fig. 4D–F). Finally, we used etoposide resistence of the cells (Suppl. Fig. 5), implying that adequate SHPRH
treatment, as inactivation of Rad5 in yeast has been shown to affect levels are required for this function of the protein.
double strand break repair [30]. Intriguingly, a reproducible effect We thus conclude that inactivation of SHPRH does not affect sen-
was detected here in different knockout clones and experiments sitivity to drugs requiring other components of the Rad6 pathway,
in case of strategy TSS (Fig. 4G) and to a lesser extend in case which is potentially due to redundancy of SHPRH with another E3
of PHD (Fig. 4H). Deletion of the RING domain alone did not ligase mediating DNA damage-induced PCNA polyubiquitination.
Fig. 3. Effects of SHPRH inactivation on PCNA ubiquitination. Induction of PCNA ubiquitination upon treatment with H2O2. (A) For targeting strategy TSS, three different
exposures are shown. (B) For targeting strategy PHD, clones were divided into two sets according to the heterozygous clone from which they were generated. Two
different blot exposures are shown for each set. (C) Two different blot exposures are shown for targeting strategy RING. ∅: untreated. Ub1: mono-ubiquitination. Ub2:
di-ubiquitination. Ub3: tri-ubiquitination. exp. = exposure.
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
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Fig. 4. Effects of SHPRH inactivation on drug survival. Clonogenic survival assays of the SHPRH targeted clones upon treatment with methyl methanesulfonate (MMS) (A–C),
−/− − K164R
cisplatin (D–F) and etoposide (G–I). In case of MMS and cisplatin treatment, Cre1 Rad18 or V PCNA cells were used as positive controls. Each point represents the
mean of 2 independent experiments (or 3 assays for etoposide treatment in TSS and PHD cells). For the etoposide assay using PHD cells, only 4 representative clones
are shown for clarity. The error bar represents the standard deviation, shown for clarity only for the positive error.
However, etoposide survival is clearly affected by SHPRH expres- Suppl. Fig. 7A). Sequencing of different representative subclones
sion. In agreement with this, SHPRH knockout cells were also also revealed no difference in the number of overall Ig gene conver-
sensitive to other double strand break inducing drugs, such sion events (Fig. 5B and C). However, the total number of nucleotide
as Doxorubicin (Suppl. Fig. 6A), but not to the UV-mimetic 4- changes due to Ig gene conversion was significantly increased in
Nitroquinoline 1-oxide (4-NQO, Suppl. Fig. 6B). the SHPRH knockout cells in a sequence analysis for two clones of
each genotype (Fig. 5D and E), which may be linked to a moder-
2.4. Influence of SHPRH on immunoglobulin diversification ate increase in Ig gene conversion tract length (Fig. 5F and G and
Suppl. Fig. 8). SHPRH might thus moderately increase the number
To assess a potential function of SHPRH in DNA double strand of tracts containing more mismatches during Ig gene conversion
break repair in a physiological context, we used the knockout strat- events, leading to more base changes even though overall event
egy TSS in DT40Cre1 cells performing Ig gene conversion. These number is not altered.
cells harbor a frameshift mutation in the rearranged Ig light chain To investigate whether SHPRH might also play a role in somatic
−
gene which may be reverted by gene conversion with upstream hypermutation, we used the DT40 V cells carrying an SHPRH
pseudogenes, thus allowing a measurement of surface IgM expres- inactivation via strategy PHD. In these cells, deletion of the pseu-
sion gain as a surrogate marker for Ig gene conversion activity [31]. dogenes required for Ig gene conversion leads to processing of
Analysis of Ig restoration in multiple individual subclones gener- AID-induced lesions by mutagenesis. The cells carry a functional
ated from the cells with the different genotypes did not reveal a Ig gene and hence surface IgM receptor, and AID-triggered delete-
substantial change in overall Ig gene conversion activity (Fig. 5A and rious mutations lead to sIgM loss as a surrogate marker of somatic
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
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Fig. 5. Influence of SHPRH on Ig gene conversion. (A) Ig gene conversion measured by sIgM restoration in subclones of the indicated genotype after 2 weeks. P-values
(Student’s t-test) are given only in case of significance. (B) Schematic illustration of the variable region of the Ig light chain. The sequenced region is indicated by an arrow
for the sequencing primer and a subsequent stretch of 698 bp. The complementary determining regions (CDR1–3) are indicated by dark gray boxes. L: leader. V: variable. J:
joining. (C) The total number of gene conversion events of representative subclones of the gene conversion assay shown in (A). The amount of analyzed sequences is indicated
below. Significance analysis: Fisher’s Exact Test. (D) Absolute number of total substitutions in the sequences shown in (C). Significance analysis: Fisher’s Exact Test. (E) The
total number of substitutions shown in (D) is classified into gene conversion mutations (GCM), ambiguous mutations (AM) and point mutations (PM). Significance analysis:
Fisher’s Exact Test. (F) The minimum (min.) length of gene conversion events as defined from the first base change to the last base change. (G) The maximum (max.) length of
Ig gene conversion events as characterized by the maximum length of homology between the analyzed sequence and the most likely pseudogene with which recombination
occurred. P-values are given (Student’s t-test) for (F and G).
hypermutation activity [32]. Analysis of surface Ig loss in multi- 3. Discussion
ple subclones generated from cells of the different genotypes did
not reveal substantial effects of SHPRH (Fig. 6A and Suppl. Fig. 7B). In the present study, we used three different strategies to inacti-
Likewise, sequencing of representative subclones did not indicate vate SHPRH, an E3 ligase implicated in PCNA polyubiquitination. As
significant changes in either mutation frequency or mutagenesis parental cells for the knockout, we used chicken DT40 B cells that
pattern in SHPRH knockout cells (Fig. 6B, C and D). We thus con- do not harbor a HLTF ortholog. The results of our study are summa-
clude that SHPRH does not play a role in somatic hypermutation rized in Table S6. We show that deletion of only the RING domain
even in cells lacking HLTF, consistent with a redundant role in DNA does not cause the same phenotype as inactivation of larger regions
damage-induced PCNA ubiquitination and base damage survival in of the SHPRH gene. The latter form of SHPRH inactivation causes
these cells. no difference in H2O2-induced PCNA polyubiquitination. Survival
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
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Fig. 6. Influence of SHPRH on Ig somatic hypermutation. (A) sIgM loss in subclones of the indicated genotypes after 2 weeks. P-values (Student’s t-test) are given in case of
significance and the number of analyzed subclones is indicated below. (B) Schematic illustration of the variable region of the Ig light chain, as given in Fig. 5B, indicating
deletion of the pseudogenes. (C) Sequence analysis of representative subclones of the Ig loss assay shown in (A). The number of point mutations per DNA sequence is given
in the periphery of the chart segments and the total number of analyzed sequences is given in the middle of the pie chart diagram. Significance analysis: Student’s t-test. (D)
The pattern of total nucleotide substitutions for each genotype is shown on the left side and the percentages of total mutations is given on the right.
in the presence of MMS and cisplatin, two drugs that greatly chal- Irrespective of this caveat, our data reveal new functions of
lenge cells lacking other components of the Rad6 pathway [29], is SHPRH in DNA repair, and add to findings supporting a high redun-
not impaired. In contrast, drugs causing DNA double strand breaks dancy of E3 ligases involved in PCNA polyubiquitination [19]. The
lead to decreased survival of SHPRH knockout cells. While the over- cells we used do not contain HLTF, i.e. no such gene was found
all frequency of Ig gene conversion is not affected, base exchanges in the chicken genome despite existent synteny at the respective
during this process, which is at least in part based on DNA dou- chromosomal position. This is interesting as such, suggesting that
ble strand break repair [33], appear to be moderately increased, even HLTF functions not related to DNA repair [41–43] became
while mutagenesis during somatic hypermutation (which does not redundant during avian evolution.
require double strand break repair [34]) is not affected. Our study Despite the lack of HLTF and different strategies for SHPRH
highlights the complexity of E3 ligase function in the Rad6 pathway, inactivation, we can detect efficient DNA damage-induced PCNA
and their contributions to other mechanisms of DNA repair. polyubiquitination in SHPRH knockout cells. Certainly, other E3
E3 ligases belong to two main functional families, RING and ligases may support this reaction in this cellular setting, strength-
HECT [35–37]. While in the HECT family, the ubiquitin is covalently ening the conclusion of an earlier report in mouse cells [19]. To date,
bound to the E3 ligase for transfer to the target protein, RING type the identity of the E3 ligase that apparently mediates most PCNA
E3 ligases employ the RING domain to foster proximity of the ubiq- polyubiquitination upon treatment with genotoxic drugs remains
uitinating E2 enzyme and its target [38–40]. As RING E3 ligases may unclear, despite a considerable and long term quest for functional
contain other binding sites for either E2 proteins or the target pro- Rad5 orthologs in vertebrates. Its identification will certainly be a
tein, and are often multidomain proteins encoded by several exons, challenge, given that most cell systems studied do contain SHPRH
their inactivation is indeed a challenge. Truncated proteins formed and HLTF, and it is unknown to which extend the ligases may really
upon alternative transcription initiation or splicing may be either replace each other.
partially functional or dominant negative. In the present study, for As another obstacle in such studies, reproducible detection of
example, we could not detect any phenotype in an SHPRH mutant PCNA polyubiquitination in normal cells is exceedingly difficult
lacking only the supposedly crucial C-terminal RING domain, while [4]. We induced highly efficient PCNA ubiquitination by H2O2 [14],
in a previous study of our lab an analogous Rad18 protein lacking as we were not able to obtain equivalent or detectable levels of
the N-terminal RING domain showed phenotypes despite in-frame PCNA polyubiquitination with other drugs (data not shown). Given
splicing over the inactivated exons [25]. Such differential effects of that the E3 ligases involved in PCNA ubiquitination may perform
the knockout strategy must be considered when interpreting data DNA damage-specific functions [4], this limitation may preclude
on RING type E3 ligase inactivation. their adequate identification. Moreover, western blotting does not
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
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detect PCNA polyubiquitination in its strict sense, but rather di- and 4. Materials and methods
triubiquitination at best. The minimum ubiquitin chain length on
PCNA required for activation of the error-free damage bypass is not Antibodies. Antibodies used for immunoblotting were: anti-AID
known. However, en bloc transfer of the ubiquitins [24] may lead (EK2 5G9, Cell Signaling), anti-HA (3F10, Roche) anti-PCNA (ab-29,
to high molecular weight modified PCNA, precluding detection of Abcam), anti-SHPRH (HPA034854, Sigma–Aldrich), anti-vinculin
polyubiquitination. (BZL03106, Biozol), anti-tubulin (ab4047, abcam) and anti-actin
In this context, it may interesting that we detect a potential (A2066, Sigma–Aldrich).
◦
slight increase in spontaneous PCNA monoubiquitination in cells Cell culture. The DT40 cell lines were cultured at 41 C and
lacking SHPRH function, despite being unable to detect polyu- 5% CO2. The RPMI 1640 medium (Invitrogen; 21875091) was
biqutination bands in their SHPRH-proficient parental cells in the supplemented with 10% FCS (Biochrom AG), 100 g/l peni-
steady state situation. While it is possible that SHPRH directly cillin/streptomycin, 2 mM glutamine, 1 mM sodium pyruvate (all
affects spontaneous PCNA monoubiquitination, this finding could GIBCO), 1% chicken serum (Sigma) and 0,1 M 2-mercaptoethanol
also suggest that a certain portion of PCNA is polyubiquitinated in (Sigma). Transfections were performed with a Gene Pulser Xcell
a high molecular weight form in normal culture cells depending on (BioRad) set at 50 F and 800 V.
SHPRH function, e.g. due to replication intermediates [44] or other Inactivation and overexpression of SHPRH in DT40 cells. The
types of spontaneous DNA damage. As both, the consequences of homologous arms of targeting vectors for the different knock-
PCNA polyubiquitination as well as clear phenotypes of its inacti- out strategies of SHPRH were amplified from genomic DT40 DNA
vation are barely known, further studies of this phenomenon will (primers: Suppl. Table 5). The PCR products were restricted using
be challenging, though. the enzymes annotated in Suppl. Table 5 and cloned into a pBlue-
In contrast to others, we do not detect a clear effect of SHPRH script II KS (+/−) vector (Fermentas). Three different loxP-flanked
inactivation on MMS survival [6]. Also, cisplatin survival that resistance cassettes (from ploxpuro, ploxbsr and ploxgpt) [49]
clearly requires the Rad6 pathway is not affected. As a note of were inserted between the arms using BamHI (strategy TSS and
caution, one should consider that redundancy in E3 ligase func- RING) or BclI (strategy PHD) restriction sites.
−
tion in the Rad6 pathway may be mediated by other proteins in In DT40 V , the first SHPRH allele was disrupted with a blas-
chicken than in mammals, as HLTF is lacking. As HLTF has been ticidin based and the second allele with a mycophenolic acid (gpt)
shown to also support PCNA monoubiquitination in one study based targeting vector. In DT40Cre1, the first SHPRH allele was
with mammalian cells [4], such considerations require careful disrupted with a puromycin based and the second allele with a blas-
choice of the right organism/cell system in the study of Rad6 path- ticidin based targeting vector. Drug resistant single cell clones were
way functions. Adding to these thoughts, expression of proteins selected with 0,8 g/ml puromycin (Sigma–Aldrich), 5 g/ml blas-
involved in the Rad6 pathway may be cell type- or organ-specific ticidin S HCl (Mobitec GmbH), 30 g/ml gpt (VWR), respectively, or
[45], presumably because not all cells should be predestined with 2 of the appropriate selections simultaneously, if necessary.
to initiate potentially mutagenic bypass of DNA damage during Drug resistant subclones were screened for targeted integration
replication. by PCR (Expand Long Template PCR Sytem, Roche) using a primer
In any case, though, our finding of defective etoposide sur- binding upstream of the homologous arm in combination with
vival in SHPRH knockout cells is interesting, as it adds to previous primers binding in the respective resistance cassette (Suppl. Table
findings implicating Rad5 function in double strand break repair 5). Gene disruption was also confirmed by southern blot analysis
[30]. In this case, excessive PCNA monoubiquitination was found using enriched cellular DNA cleaved with BclI for strategy TSS
to interfere with homologous recombination. We also detected and PHD or PvuII for strategy RING. The different probes were
defective etoposide survival in the mutants apparently showing amplified from genomic DT40 DNA (primers: Suppl. Table 5) and
◦
a moderately higher spontaneous PCNA monoubiquitination. As the membrane was incubated with the particular probe at 65 C.
a potential mechanistic approach to decipher this phenomenon, Recycling of the loxP-flanked selectable marker gene was
we see higher tolerance for mismatches during Ig gene conver- achieved by overnight culture in 1 M 4-hydroxy tamoxifen
sion in SHPRH knockout cells. PCNA ubiquitination has not been (H7904, SIGMA), followed by limiting dilution subcloning [49].
implicated in general mismatch repair, but PCNA monoubiquitina- For overexpression, the coding sequence of DT40 SHPRH was
tion was shown to induce non-canonical mismatch repair functions amplified and tagged with HA on the N-terminus (primers: Suppl.
that may contribute to mutagenesis [13]. It is tempting to specu- Table 5) using Phusion polymerase (Finnzymes) and cut with EcoRV
late that mismatch recognition or exclusion during homologous at the 5 - and 3 end for cloning into pExpress [49]. Subsequently,
recombination could be impaired in SHPRH knockout cells. One the cDNA expression cassette was excised with SpeI and cloned into
must consider, though, that intrinsic mismatch tolerance is quite the NheI site of ploxpuro.
high in DT40 cells, as several mismatches are tolerated in even For RT-PCR and quantitative RT-PCR (qRT-PCR), RNA was iso-
short conversion tracts as well as in gene targeting approaches lated (RNeasy Mini Kit, Qiagen) and reversely transcribed using
[46,47]. It will thus be interesting to investigate in a mammalian the First Strand cDNA Synthesis Kit (Roche). For RT-PCR, primers
cell system whether the fidelity of homologous recombination amplifying from exon 4 to 8 and exons 25 to 28 were used (Suppl.
is affected upon inactivation of SHPRH or rather PCNA polyu- Table 5). For qRT-PCR, primers amplifying from exons 25 to 28 and
biquitination, once the technical obstacles described above are amplifying the glyceraldehyde 3-phosphate dehydrogenase gene
overcome. (GAPDH) were used (Suppl. Table 5). The expression levels of the
Our study reveals a considerable complexity in the analysis of housekeeping gene GAPDH serve as control to determine relative
E3 ligases involved in PCNA ubiquitination and other functions. SHPRH expression levels.
However, it is worthwhile considering how and why redundancy in Analysis of drug survival and immunoblot analysis. A colony-
these enzymes may have evolved, given that our genome encodes forming assay was used to analyze the survival in the presence
for over 600 E3 ligases [48] to ubiquitinate most cellular proteins. of etoposide (Sigma, USA), doxorubicin (Santa Cruz, Germany),
Similarly to phosphorylation, targeted ubiquitination is a versatile cisplatin (Ribosepharm, Gräfelfing) or methyl methanesulfonate
tool to change protein function. Hence, triggering modification of (MMS) (Sigma, USA) as previously described [50]. In short, sensi-
the same lysine residue by more than one pathway, employing tivity to the drugs was measured plating cells onto methylcellulose
more than one E3 ligase, may facilitate to adequately exploit its containing the drug, and colonies were counted after 10–14 days
full potential in cellular regulation. of culture. PCNA mono- and polyubiquitination was induced by
Please cite this article in press as: N.-S. Tomi, et al., Analysis of SHPRH functions in DNA repair and immunoglobulin diversification, DNA
Repair (2014), http://dx.doi.org/10.1016/j.dnarep.2014.09.010
G Model
DNAREP-2005; No. of Pages 10 ARTICLE IN PRESS
N.-S. Tomi et al. / DNA Repair xxx (2014) xxx–xxx 9
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This work was supported by the Deutsche Forschungsgemeinschaft
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(JU2690/1-2 and a Heisenberg fellowship to B.J.).
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Repair (2014), http://dx.doi.org/10.1016/j.dnarep.2014.09.010