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

Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
DNA repair (Impact Factor: 3.11). 04/2011; 10(4):438-44. DOI: 10.1016/j.dnarep.2010.12.008
Source: PubMed


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).

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Available from: Heinz Jacobs, Oct 03, 2015
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    • "Wild-type human lymphocytes [49] were cultured in RPMI supplemented with 10% FBS, 1 mM L-glutamine, 1 mM sodium pyruvate (Life Technologies), 1% non-essential amino acids (Life Technologies), 50 µM 2-mercaptoethanol and 50 µg/ml gentamycin sulfate (Life Technologies). Wild-type and SHPRH mouse embryonic fibroblasts (MEFs) [50], RPE (ATCC), HEK293T (human embryonic kidney) (ATCC), HeLa (human cervical adenocarcinoma) (ATCC), HCT116 (human colorectal cancer cell) (ATCC), U2OS EJ2-GFP [40], isogenic HCT116 FANCM- [43], [46] and LIG4- [43], [44] null cells, HCC1937 cells complimented with a BRCA1 cDNA and HCC1937 [45] were cultured in DMEM with Glutamax (Life Technologies) supplemented with 10% FBS and 1% penicillin- streptomycin. U2OS DR-GFP [34], [51] cells were cultured in McCoys modified 5A (Life Technologies) supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin. "
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    • "In this scenario, HLTF could promote the error-free bypass of the lesions by promoting D-loop formation. On the other hand, mice cells lacking HLTF are as resistant as wild-type cells to a variety of DNA damaging agents, suggesting a functional overlap with either other functional homologue of Rad5, SHPRH or other repair pathways (46,47). Clearly, more experiments will be required to understand the role of HLTF in genome stability as well as cooperativity and regulation of parallel repair pathways. "
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