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Analysis of SHPRH Functions in DNA Repair and Immunoglobulin Diversification

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Analysis of SHPRH Functions in DNA Repair and Immunoglobulin Diversification G Model DNAREP-2005; No. of Pages 10 ARTICLE IN PRESS DNA Repair xxx (2014) xxx–xxx 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 Repair (2014), http://dx.doi.org/10.1016/j.dnarep.2014.09.010 G Model DNAREP-2005; No. of Pages 10 ARTICLE IN PRESS 2 N.-S. Tomi et al. / DNA Repair xxx (2014) xxx–xxx 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 DT40V 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 DT40V 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.
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