HLTF and SHPRH are not essential for PCNA polyubiquitination, survival and somatic hypermutation: existence of an alternative E3 ligase

Abstract

DNA damage tolerance is regulated at least in part at the level of proliferating cell nuclear antigen (PCNA) ubiquitination. Monoubiquitination (PCNA-Ub) at lysine residue 164 (K164) stimulates error-prone translesion synthesis (TLS), Rad5-dependent polyubiquitination (PCNA-Ub(n)) stimulates error-free template switching (TS). To generate high affinity antibodies by somatic hypermutation (SHM), B cells profit from error-prone TLS polymerases. Consistent with the role of PCNA-Ub in stimulating TLS, hypermutated B cells of PCNA(K164R) mutant mice display a defect in generating selective point mutations. Two Rad5 orthologs, HLTF and SHPRH have been identified as alternative E3 ligases generating PCNA-Ub(n) in mammals. As PCNA-Ub and PCNA-Ub(n) both make use of K164, error-free PCNA-Ub(n)-dependent TS may suppress error-prone PCNA-Ub-dependent TLS. To determine a regulatory role of Shprh and Hltf in SHM, we generated Shprh/Hltf double mutant mice. Interestingly, while the formation of PCNA-Ub and PCNA-Ub(n) is prohibited in PCNA(K164R) MEFs, the formation of PCNA-Ub(n) is not abolished in Shprh/Hltf mutant MEFs. In line with these observations Shprh/Hltf double mutant B cells were not hypersensitive to DNA damage. Furthermore, SHM was normal in Shprh/Hltf mutant B cells. These data suggest the existence of an alternative E3 ligase in the generation of PCNA-Ub(n).

Fig. 1

Role of the Rad6 epistasis group in SHM. The ring-shaped PCNA homotrimer encircles DNA, and tethers DNA polymerases to the DNA template. Rad6/Rad18 monoubiquitinates PCNA at lysine 164 (K164). This modification is thought to recruit the TLS polymerase η to generate mutations at template A/T. Further K63-linked polyubiquitination of PCNA-Ub by Ubc13/Mms2 and SHPRH or HLTF activates template switching (TS) and may (?) suppress PCNA-Ub-dependent TLS, i.e. A/T mutagenesis during SHM. Furthermore, PCNA-Ubⁿ may stimulate TLS polymerases and affect SHM.

Fig. 2

Generation of Shprh-mutant mice. (A) Shprh was inactivated in the mouse germ line by making use of an exon-trapped ES cell clone. As indicated schematically, this clone carries a lacZ/Neo^R exon in intron 16 of mouse Shprh. The location of the three different primer sets (P1–3) used in (D), are indicated. (B) The Shprh mutation generates a ΔShprh-βgal-neo fusion in which the C-terminal portion of SHPRH harboring the RING and helicase domain of SHPRH are replaced by βgal-Neo. The location of the three different primer sets (P1–3) used in (D), are indicated. (C) As revealed by Western blotting, the exon trap strategy only allows the generation of a single mutant protein species, which is consistent with the location of the exon trap and compared to the wild type SHPRH protein has lower mobility. (D) Expression of exon 17 (P1) and the RING and helicase domain (P3) of shprh gene, and the lacZ/Neo^R exon (P2) revealed by RT-PCR with Actin transcript level as a loading control. (E) mRNA Levels of the RING domain of Hltf were determined by quantitative real-time PCR in stimulated B cells and related to mRNA levels of Hprt. Data are means of 2 independent reactions.

Fig. 3

PCNA Polyubiquitination. (A) MEFs from wild type, Shprh/Hltf-deficient, and PCNA^K164R mice were irradiated with 60 J/m² UV-C and incubated 12 h at 37 °C. After isolating chromatin bound fraction from the cells, immunoprecipitations were carried out using an anti-PCNA antibody. The immunoprecipitated proteins were analyzed by immunoblotting with the anti-PCNA antibody to detect the presence of the PCNA ubiquitinaiton. *A protein species of unknown identity. (B) Short exposure of the unmodified PCNA band.

Fig. 4

Surival and Class Switch Recombination. Shprh/Hltf-deficient (black) and wild-type (grey) B cells were stimulated with LPS and exposed to increasing doses of either cisplatin (a), UV-C (b), or MMS (c). The percentage of survival after four days of culture is shown. Comparison of wild-type (grey) and Shprh/Hltf double-deficient B cells (black bars) switched to IgG3 upon activation by LPS, or switched to IgG1 upon activation by LPS and IL-4 (d).

Fig. 5

Mutations in rearranged J_H4 intronic sequences from wild type, PCNA^K164R, ΔShprh/Hltf mutant and Shprh/Hltf-deficient B cells. (A) Pattern of nucleotide substitution. Values are expressed as the total number of mutations and percentage of total mutations. (B) Relative contribution of A/T mutations, G/C transitions (TRS) and G/C transversions (TRV). Values are expressed as the percentage of total mutations.