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Generation of a Mono‐ubiquitinated PCNA Mimic by Click Chemistry
Author(s) -
Eger Silvia,
Castrec Benoît,
Hübscher Ulrich,
Scheffner Martin,
Rubini Marina,
Marx Andreas
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100444
Subject(s) - proliferating cell nuclear antigen , processivity , dna polymerase , dna clamp , dna polymerase delta , dna replication , ubiquitin , polymerase , dna , chemistry , microbiology and biotechnology , dna polymerase ii , dna synthesis , replication factor c , biochemistry , biology , eukaryotic dna replication , polymerase chain reaction , reverse transcriptase , gene
Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high‐fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono‐ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site‐specifically mono‐ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site‐specifically by the Cu I ‐catalyzed azide–alkyne cycloaddition. Finally, we show that the synthetic PCNA–Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA–Ub than to PCNA.