title: Analysis of SHPRH functions in DNA repair and immunoglobulin diversification.
abstract: During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH 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 hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs 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.
authors.affiliation: Department of Cell Biology, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich-Schiller University Jena, Hans-Knoell-Strasse 2, 07745 Jena, Germany.
authors.forename: Nils-Sebastian
authors.initials: NS
authors.lastname: Tomi
authors.affiliation: Department of Cell Biology, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich-Schiller University Jena, Hans-Knoell-Strasse 2, 07745 Jena, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Davari
authors.affiliation: Institute of Clinical and Molecular Biology, Helmholtz Center Munich, Marchioninistrasse 25, 81377 Munich, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Grotzky
authors.affiliation: Institute of Clinical and Molecular Biology, Helmholtz Center Munich, Marchioninistrasse 25, 81377 Munich, Germany.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Loos
authors.affiliation: Department of Cell Biology, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich-Schiller University Jena, Hans-Knoell-Strasse 2, 07745 Jena, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Böttcher
authors.affiliation: Institute of Clinical and Molecular Biology, Helmholtz Center Munich, Marchioninistrasse 25, 81377 Munich, Germany.
authors.forename: Samantha
authors.initials: S
authors.lastname: Frankenberger
authors.affiliation: Department of Cell Biology, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich-Schiller University Jena, Hans-Knoell-Strasse 2, 07745 Jena, Germany. Electronic address: berit.jungnickel@uni-jena.de.
authors.forename: Berit
authors.initials: B
authors.lastname: Jungnickel
mesh_terms: D000818:Animals; D002460:Cell Line / Q000187:drug effects; D002645:Chickens; D002945:Cisplatin / Q000494:pharmacology; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D005047:Etoposide / Q000494:pharmacology; D055786:Gene Knockout Techniques; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D007136:Immunoglobulins; D018809:Proliferating Cell Nuclear Antigen; D005047:Etoposide; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D002945:Cisplatin
keywords: DNA repair; DT40; PCNA; SHPRH; Ubiquitin