Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Motegi, Akira; Sood, Raman; Moinova, Helen; Markowitz, Sanford D.; Liu, Pu Paul; Myung, Kyungjae

doi:10.1083/jcb.200606145

Cited by 180 publications

(188 citation statements)

References 23 publications

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“…On the other hand, SHPRH has an H1 and H5 linker sequence and a PHD domain not present in either Rad5 or HLTF. Because SHPRH can also promote the polyubiquitination of PCNA (12,13), it will be important to examine the overlaps and differences between the functions of HLTF and SHPRH, suggested by their domain structures.…”

Section: Discussionmentioning

confidence: 99%

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Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

Unk

Hajdú

Fátyol

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5⌬ yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6 -Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancersuppression role.yeast Rad5 ͉ damage bypass ͉ K63 polyubiquitination ͉ tumor suppressor L esions in DNA impose a block to synthesis by the replicative polymerases (Pols), and unless replication is rescued by the timely action of lesion bypass processes, stalled replication forks can collapse, leading to genomic instability. In eukaryotes the Rad6-Rad18 enzyme complex regulates lesion bypass processes that ensure the completion of replication. Rad6, a ubiquitinconjugating enzyme, forms a tight complex with Rad18, a RING-finger type ubiquitin ligase that binds DNA (1, 2), and in cells treated with DNA-damaging agents, Rad6-Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA), a DNA Pol sliding clamp that is a key component of the replication machinery (3). Ubiquitination at the Lys-164 residue of PCNA and its subsequent polyubiquitination serves as a molecular switch between various DNA damage bypass processes (3-5).In the yeast Saccharomyces cerevisiae, Rad6-Rad18 governs at least three alternative pathways for promoting replication through DNA lesions (6). Two pathways activated by PCNA monoubiquitination are carried out by specialized translesion synthesis (TLS) DNA Pols, such as Pol and Pol , which are able to copy DNA directly from the damaged template on an error-free or error-prone way (6). The third pathway called postreplication repair (PRR), however, is activated by PCNA polyubiquitination and operates by template switching using the information of the undamaged newly synthesized nascent strand on the sister duplex for DNA synthesis across damaged DNA (7-10). The PRR pathway depends on the Rad5, Mms2, and Ubc13 proteins and promotes error-free replication through DNA lesions. Recently, we have shown that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression, as Rad5 can concertedly unwind and anneal the nascent and the parental strands of the fork without exposing any single-stranded regions (7). This Rad5 activity would ensure damage bypass by promoting replication fork regression where the newly synthesized DNA strand of the sister duplex can be used as a template. In addition to its r...

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Section: Discussionmentioning

confidence: 99%

“…Previously, we and others have shown that human SHPRH, which resembles Rad5 in having a SWI/SNF helicase domain and a C 3 HC 4 RING motif, exhibits a ubiquitin ligase activity able to promote PCNA polyubiquitination in a Rad6-Rad18-and Mms2-Ubc13-dependent manner (12,13).…”

mentioning

confidence: 94%

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

Unk

Hajdú

Fátyol

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

221

224

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“…SHPRH directly interacts with PCNA, RAD18, and UBC13, but not with RAD6 (9). To examine whether HLTF has similar binding properties, 3XFLAG-HLTF-containing cell extract was incubated with GST-fused PCNA, UBC13, or RAD6 ( Fig.…”

Section: Physical Interactions Between Hltf and Proteins Participatinmentioning

confidence: 99%

“…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)(9)(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.…”

mentioning

confidence: 99%

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

Motegi

Liaw

Lee

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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251

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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...

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“…Regardless of the mode of reaction, it is abundantly clear that the error-free DDT process is highly conserved in the entire eukaryotic kingdom, from yeast to human. Sequence and functional homologs of all proteins involved in error-free DDT, including Mms2 [140], Ubc13 [141] and Rad5 [142,143], have been found in mammals, plants and other higher eukaryotes [144,145]. For a few limited examples, suppression of the above genes resulted in phenotypes reminiscent of the corresponding yeast mutants [143,146,147].…”

Section: Error-free Ddtmentioning

confidence: 99%

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Andersen¹,

Xiao³

2007

Cell Res

185

187

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npg In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer. DNA damage toleranceIn the presence of spontaneous or carcinogen-induced DNA damage, living cells have to maintain and complete DNA synthesis or risk replication fork collapse. Since the process of DNA licensing is to ensure the genome is duplicated once and only once during each cell cycle, stalled or collapsed replication forks may not be able to restart, which often results in double-strand breaks (DSBs) and causes compromised genome integrity or cell death. In addition to highly conserved DNA repair pathways, all living organisms have evolved schemes to ensure continuation of DNA synthesis in the presence of damage. These schemes were originally termed DNA postreplication repair (PRR) due to observations of transient shortened nascent DNA structures following S phase in response to DNA damage. In bacteria and unicellular yeast, these shortened DNA segments can be measured by an alkaline sedimentation assay [1] or directly observed in electron micrographs [2]. In wild-type cells, these truncated DNA segments were restored to full length following a short recovery time. One typical experiment [1] involved the restoration of the nascent strand following UV exposure in nucleotide excision repair (NER)-deficient cells and was originally assumed to represent a mechanism of DNA repair. However, further investigation revealed that, although the nascent fragments were re-annealed, the original UV-induced pyrimidine dimers, which were responsible for the generation of single-strand gaps, often persisted in the genome [3,4]. It was argued that the replication-blocking lesion was not necessarily corrected, but rather transiently bypassed and carried over to the next generation. Perhaps it is more beneficial for the organi...

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Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Cited by 180 publications

References 23 publications

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

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

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

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