Abstract
Proliferating cell nuclear antigen (PCNA) is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNAK164R mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.
Highlights
Proliferating cell nuclear antigen (PCNA), a homotrimeric DNA-encircling protein, is the key target of the conserved ubiquitin-dependent RAD6 pathway of post-replicative DNA repair [1]
Studies in the yeast Saccharomyces cerevisiae suggest that the switch from replicative to translesion DNA synthesis is mediated by PCNA ubiquitination catalyzed by the E2 ubiquitin-conjugating enzyme Rad6 and the E3 ubiquitin ligase Rad18 [1,2]
The results indicate that the PCNAK164R mutation prevents PCNA ubiquitination and SUMOylation, whereas the RAD18 gene disruption decreases, but does not abolish mono-ubiquitination of PCNA in DT40
Summary
Proliferating cell nuclear antigen (PCNA), a homotrimeric DNA-encircling protein, is the key target of the conserved ubiquitin-dependent RAD6 pathway of post-replicative DNA repair [1]. Studies in the yeast Saccharomyces cerevisiae suggest that the switch from replicative to translesion DNA synthesis is mediated by PCNA ubiquitination catalyzed by the E2 ubiquitin-conjugating enzyme Rad and the E3 ubiquitin ligase Rad18 [1,2]. Mono-ubiquitination of human PCNA requires the human Rad homologue and increases the affinity of PCNA for the translesion DNA polymerases Polg [3,4] and REV1 [5]. Studies in S. cerevisiae have shown that the lysine-to-arginine substitution at amino acid position 164 of PCNA (PCNAK164R) prevents ubiquitination, but does not interfere with the essential function of PCNA in replication [1]. K164 is the target of RAD18-mediated PCNA ubiquitination in higher eukaryotes [1,3]
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