Synthesis of Free and Proliferating Cell Nuclear Antigen-bound Polyubiquitin Chains by the RING E3 Ubiquitin Ligase Rad5

Abstract

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes errorfree bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys 63 -linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13ϳUb thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys 63 of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.DNA is susceptible to chemical alteration by many endogenous and exogenous agents. To counter this threat and maintain genome integrity, eukaryotic cells employ three main strategies: DNA repair pathways that directly reverse DNA damage, cell cycle checkpoints that allow time to repair the damage prior to replication, and DNA damage tolerance (DDT), 2 which is a method of bypassing DNA damage lesions during the DNA replication phase of the cell cycle.Proliferating cell nuclear antigen (PCNA) is a key regulatory protein in DNA replication and repair (1). At the replication fork, DNA is encircled by PCNA, a homotrimeric protein that promotes processive movement of the replicative DNA polymerase. Upon DNA damage and subsequent stalling of the replicative polymerase, Ub modifications of PCNA signal DDT, which allows a cell to bypass the lesion and proceed past this potential block in replication (2-4).In the DDT pathway, as in other Ub-dependent pathways, Ub is conjugated to a substrate by the actions of three enzymes, an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase (5). The E1 enzyme initiates the pathway in a two-step reaction that utilizes ATP hydrolysis to activate the C terminus of Ub, culminating in the formation of an E1ϳUb thiolester. Subsequent transthiolation to the active site cysteine of the E2 generates an E2ϳUb thiolester. An E3 ligase then brings a substrate into close proximity to the E2ϳUb intermediate, thereby catalyzing the formation of an isopeptide bond between the amino group of a substrate lysine and the C-terminal glycine of Ub. Polyubiquitination occurs when this substrate is another Ub, either free or as part of a Ub-protein conjugate.The DDT pathway is characterized by distinct ubiquitination events on PCNA that occur in two stages (3,4,6). T...

Generation of nonhydrolyzable ubiquitin–histone mimics

“…Several mimics of the native isopeptide bond have been introduced that are resistant to DUB cleavage, such as oxime or triazole linkages [24–26]. The approach we used to generate nonhydrolyzable Ub-histone mimics was derived from a strategy previously employed to generate diUb [27] and ubiquitinated PCNA [28] analogs. It takes advantage of a highly reactive bifunctional thiol crosslinker, 1,3-dichloroacetone.…”

Error-free DNA-damage tolerance in Saccharomyces cerevisiae

“…Since this lesion bypass mechanism could be activated simply by the recruitment of Rad5 to a stalled replication fork in the presence of Rad18 and monoubiquitinated PCNA [30,45], the replication fork regression model cannot explain the requirement of PCNA polyubiquitination for error-free DDT. It remains possible though that polyubiquitinated PCNA functions to inhibit the association or the assembly of unwanted factors at the damaged site that might otherwise prevent replication fork regression.…”

“…Both of these activities are essential for functional error-free DDT as mutations in the associated domains impart sensitivity to DNA damage and increased spontaneous mutagenesis similar to an mms2 or ubc13 mutant [43]. In addition to its enzymatic activities, Rad5 physically interacts with Rad18, Ubc13 and both ubiquitinated and unmodified PCNA [30,44,45]. It appears that association of the Rad6-Rad18 E2-E3 complex with PCNA at sites of DNA damage recruits Ubc13-Mms2 through Rad5, and this second E2-E3 complex mediates polyubiquitination of the sliding clamp [18,46].…”

RAD5a and REV3 function in two alternative pathways of DNA-damage tolerance in Arabidopsis

“…Although the biological function of HIRAN domain remains unknown, based on in silico analysis, it has been described as a DNA-binding domain for recognizing damaged DNA or a stalled replication fork (Iyer et al, 2006). The finding of monoubiquitinated PCNA and Rad18 interacting with Rad5 through N-terminal half of the Rad5 HIRAN domain suggests that this domain may function as a platform for protein-protein interactions (Carlile et al, 2009;Ulrich and Jentsch, 2000). SHPRH has a PHD domain absent in Rad5 and HLTF.…”