Link: University of Iowa

Structure of Monoubiquitylated PCNA

The proliferating cell nuclear antigen (PCNA) is a trimeric, ring-shaped protein that slides along DNA to promote replication. When DNA is damaged, a replicative polymerase is replaced by a translesion polymerase in a process that involves addition of a single ubiquitin modification to each PCNA monomer. How this modification changes the appearance and function of PCNA was not understood until Todd Washington’s laboratory developed an ingenious trick.

Lacking a way to produce the endogenous monoubiquitylated PCNA in quantities suitable for structural characterization, graduate student Bret Freudenthal and associate professor Todd Washington engineered “split PCNA” constructs in which the N-terminal 163 amino acids of PCNA and the ubiquitin-linked C-terminal remainder of PCNA are co-expressed in E. coli, where they self assemble. ┬áBecause E. coli does not have the ability to add ubiquitin to a lysine sidechain, the investigators developed a way to link ubiquitin to the C-terminal piece of PCNA in a manner so nearly native that when the constructs were expressed in yeast, cells displayed normal growth and resistance to ultraviolet light.

Finally, when Freudenthal and Washington crystallized split PCNA and split monoubiquitylated PCNA, they were able to determine how addition of ubiquitin alters the structure of the PCNA trimer. Their structures revealed that ubiquitin is added to the “back” side of the PCNA trimer in a manner might allow different polymerases to be deployed from what has been termed a sliding clamp tool belt. Drs. Lokesh Gakhar and Subramanian Ramaswamy were co-authors. The paper appears in Nature Structural and Molecular Biology.

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