Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Abstract

C hronic stalling of DNA replication forks by DNA damage such as UV irradiation, ionizing irradiation, chemicals, and reactive cellular metabolites impedes the progression of the cell cycle and eventually causes cell death. To circumvent such situations, cells have evolved the postreplication repair (PRR) pathway that bypasses DNA lesions to resolve stalled forks without removing the actual damage (1). In budding yeast Saccharomyces cerevisiae, PRR is carried out by 2 distinct pathways: translesion synthesis (TLS) and template switching (TS) (Fig. 1A). TLS uses multiple low-fidelity TLS polymerases to incorporate nucleotides across DNA lesions (2, 3). Switching from replicative polymerases ␦ or to TLS polymerases is promoted through the interaction between monoubiquitinated PCNA at lysine 164 (K164) and a ubiquitin-binding motif in TLS polymerases-a mechanism conserved from the budding yeast to human. The monoubiquitination of PCNA at K164 requires the RING-type ubiquitin ligase Rad18 (E3) and the ubiquitinconjugating enzyme Rad6 (E2).The TS pathway bypasses DNA damage by switching a stalled replicating end to the nascent daughter strand of the sister chromatid (1, 4). This pathway involves a lysine 63 (K63)-linked polyubiquitin chain that is further added onto the monoubiquitinated PCNA by Rad5 (E3) along with the Ubc13-Mms2 (E2 and E2 variant, respectively) heterodimer complex (Fig. 1 A). Distinct from the K48-linked polyubiquitination leading to protein degradation, the K63-linked polyubiquitination of PCNA is thought to promote TS in a proteasome-independent manner (5).The importance of the TLS pathway in the suppression of mammalian tumorigenesis emerged with the identification of a mutation in TLS polymerase in patients with the variant form of xeroderma pigmentosum and from studies with mouse models (6, 7). Despite the presence of UBC13 and MMS2 homologues in humans, the importance of the TS pathway is less clear in mammals because K63-linked polyubiquitination of PCNA, a hallmark event for the TS pathway, had not been observed until recently (8-10). We recently identified human SHPRH, which possesses SWI2/SNF2 and RING domains with similar architecture to the yeast Rad5 as a functional homologue of yeast Rad5 (9). Specifically, we demonstrated the in vivo activity of SHPRH in promoting a K63-linked polyubiquitination of PCNA as well as physical interactions of SHPRH with PCNA, RAD18, and UBC13. Depletion of SHPRH increases genomic instability after genotoxic stress. Consistent with our work, another study also demonstrated that SHPRH could polyubiquitinate PCNA in vitro (11).In the present study, we demonstrated that ectopic expression of HLTF/SMARCA3/RUSH/HIP116/Zbu1 (hereafter, HLTF) enhanced PCNA polyubiquitination in vivo. Depletion of SHPRH or HLTF significantly reduced polyubiquitination of chromatin-bound PCNA upon treatment of cells with DNA-damaging agents that cause stalled DNA replication forks. Furthermore, Hltf-deficient mouse embryonic fibroblasts (MEFs) showed elevated chromosome breaks an...

SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner

“…SHPRH and HLTF, homologs of yeast Rad5, were previously identified as E3 ubiquitin ligases that catalyze the polyubiquitylation of PCNA and participate in methyl methane sulfonate (MMS)-induced DNA damage repair (26)(27)(28). In contrast to yeast in which Rad5-deficiency causes high sensitivity to DNA-damaging agents, silencing expression of SHPRH or HLTF in human cells or knockouts of SHPRH and HLTF in mice showed mild or no severe sensitivity to DNA damaging agents, respectively (29)(30)(31) (Fig.…”

“…RAD5 and its mammalian homolog SHPRH polyubiquitylate proliferating cell nuclear antigen (PCNA) to promote DNA damage bypass via an uncharacterized recombination-dependent pathway (24,25). Furthermore, there are two RAD5 homologs in mammals, which suggests that functions other than DNA damage bypass might have been developed during evolution (26)(27)(28).…”

BRCA1promotes the ubiquitination of PCNA and recruitment of translesion polymerases in response to replication blockade

“…Both branches of the DDT pathways appear to be more complex in mammals. For example, two Saccharomyces cerevisiae Rad5 homologs have been identified in mammals, HLTF and SHPRH (19)(20)(21)(22). Both can bind the E2 heterodimer, UBC13 and MMS2, and promote the polyubiquitination of PCNA in vitro and in vivo.…”

Proliferating Cell Nuclear Antigen (PCNA)-binding Protein C1orf124 Is a Regulator of Translesion Synthesis

“…Rad18 mediates the monoubiquitination of PCNA, whereas Rad5 facilitates the further addition of Lys-63-linked polyubiquitin chains (5,11,12). In humans, monoubiquitination on Lys-164 is the major mod-ification of PCNA detected upon exposure of replicating cells to DNA damage induced by UV light or hydroxyurea (HU) (13), whereas polyubiquitination of PCNA has recently been detected at much lower levels (14). Monoubiquitination of PCNA increases its affinity for TLS pol and pol and Rev1 (11,13,15,16).…”