Affinage

SHPRH

E3 ubiquitin-protein ligase SHPRH · UniProt Q149N8

Length
1683 aa
Mass
193.1 kDa
Annotated
2026-06-10
29 papers in source corpus 16 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

SHPRH is a SWI2/SNF2-family E3 ubiquitin ligase and human RAD5 ortholog that operates in error-free DNA damage tolerance by catalyzing K63-linked polyubiquitination of PCNA at Lys164, thereby channeling stalled replication into the error-free postreplication-repair branch (PMID:17108083, PMID:17130289). It assembles with the Rad6-Rad18 and Mms2-Ubc13 complexes and binds PCNA to drive MMS-induced PCNA polyubiquitination, with its loss increasing alkylation sensitivity and genomic instability (PMID:17108083, PMID:17130289). SHPRH and its paralog HLTF act in a damage-specific division of labor: MMS triggers HLTF degradation and licenses SHPRH-Rad18-polymerase κ error-free bypass, whereas UV favors HLTF-dependent translesion synthesis (PMID:21396873). SHPRH directly ubiquitinates Lys164 of both free and DNA-bound PCNA using either Ubc13-Mms2 or the Ube2D E2 family, an activity that requires its HIRAN domain for PCNA engagement (PMID:41990034). Biochemically it is a nucleosome-stimulated ATPase that binds the H2A-H2B acidic patch and extranucleosomal DNA through a distinct SF2 ATPase engagement mode, and acts as a promiscuous nucleosome E3 ligase capable of branched and degradation-associated ubiquitin linkages (PMID:31434570, PMID:38979307). Beyond replication-fork bypass, SHPRH localizes through its PHD domain to nucleolar rDNA promoters, where it interacts with RNA Polymerase I to support mTOR-dependent rRNA transcription (PMID:28400511), is recruited to bulky-adduct damage sites by TonEBP that sequentially coordinates SHPRH and the deubiquitinase USP1 (PMID:31376680), and additionally engages the mismatch-repair protein MLH1 and ubiquitinates the kinase FYN (PMID:35784486, PMID:34606908).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 2006 High

    Established that human SHPRH is the functional RAD5 ortholog providing the missing E3 activity for the error-free branch of postreplication repair, answering how K63-linked PCNA polyubiquitination is achieved in human cells.

    Evidence Co-IP with Rad6-Rad18 and Mms2-Ubc13, in vitro ubiquitylation and active-site analysis, plus reciprocal Co-IP with knockdown phenotypes in a parallel study

    PMID:17108083 PMID:17130289

    Open questions at the time
    • Did not define which domain mediates PCNA recognition
    • Did not resolve substrate specificity versus other E3s
  2. 2008 High

    Showed SHPRH is not the sole RAD5 homolog, revealing HLTF as a cooperating E3 in PCNA polyubiquitination and raising the question of functional redundancy versus specialization.

    Evidence Co-IP of HLTF with UBC13/PCNA/SHPRH, siRNA knockdown, mutagenesis and MEF chromosome-break assays

    PMID:18719106

    Open questions at the time
    • Mechanism distinguishing SHPRH from HLTF unresolved at this stage
  3. 2011 High

    Resolved how the two RAD5 homologs are coordinated, demonstrating damage-type-specific partitioning of postreplication-repair branches, and showed an additional E3 must exist for PCNA polyubiquitination.

    Evidence Co-IP, siRNA, TLS-polymerase recruitment and mutagenesis assays; independently, Shprh/Hltf double-knockout MEFs retaining PCNA-Ubn

    PMID:21269891 PMID:21396873

    Open questions at the time
    • Identity of the alternative PCNA E3 ligase not determined
    • Molecular trigger for damage-specific HLTF degradation not fully defined
  4. 2013 High

    Determined the SHPRH PHD-domain fold and found it does not read H3K4-methylated peptides, redirecting the search for the domain's function.

    Evidence Solution NMR structure and NMR titrations with H3 peptides

    PMID:23907177

    Open questions at the time
    • Positive binding partner of the PHD domain not identified in this study
  5. 2017 High

    Uncovered a replication-independent nucleolar role, placing SHPRH at rDNA promoters to support mTOR-dependent rRNA transcription and assigning a function to its PHD domain.

    Evidence ChIP, Co-IP with RNA Pol I, shRNA, and pharmacological inhibition (rapamycin, actinomycin D)

    PMID:28400511

    Open questions at the time
    • Whether E3 ligase activity is required for rRNA transcription not established
    • Direct substrate at the rDNA promoter not identified
  6. 2019 High

    Defined SHPRH's intrinsic biochemistry as a nucleosome-stimulated ATPase and broad nucleosome E3 ligase, indicating activity extends beyond PCNA and the Ubc13-Mms2 pair.

    Evidence Reconstituted ATPase and nucleosome-binding assays, 26-E2 screen, in vitro ubiquitylation and MS of ubiquitin linkages

    PMID:31434570

    Open questions at the time
    • Physiological nucleosomal substrates not identified
    • In vivo relevance of branched linkages not tested
  7. 2019 Medium

    Identified an upstream recruiter, TonEBP, that sequentially delivers SHPRH and then USP1 to bulky-adduct lesions, explaining how PCNA ubiquitination dynamics are timed.

    Evidence Co-IP, laser micro-irradiation live-cell recruitment, siRNA, PCNA-ubiquitination immunoblot

    PMID:31376680

    Open questions at the time
    • Single lab; reciprocal structural mapping of the SHPRH-TonEBP interface limited
    • How the SHPRH-to-USP1 switch is triggered unresolved
  8. 2020 Medium

    Linked SHPRH nuclear localization to a PCNA-interacting APIM motif and connected it to the CHK2 damage-response branch, broadening its regulatory reach.

    Evidence APIM mutagenesis with localization and SupF mutation-spectrum assays; CRISPR knockout with CHK2/MCM2 phosphorylation immunoblots

    PMID:31973093 PMID:32192191

    Open questions at the time
    • How APIM and HIRAN-mediated PCNA contacts are coordinated unclear
    • Whether CHK2 effect is direct or downstream of PCNA ubiquitination not resolved
  9. 2021 Medium

    Extended SHPRH substrates and regulation beyond replication, showing KMT2B drives SHPRH expression and SHPRH ubiquitinates FYN for degradation.

    Evidence ChIP for H3K4me3 at the SHPRH promoter, Co-IP, ubiquitination assays and overexpression/knockdown

    PMID:34606908

    Open questions at the time
    • Direct versus indirect ubiquitination of FYN not fully separated
    • Single lab
  10. 2022 Medium

    Demonstrated a conserved SHPRH-MLH1 interaction via a Rad5-derived MIP-box motif, implicating SHPRH in mismatch-repair-associated drug responses.

    Evidence Co-IP, yeast two-hybrid, bioinformatic MIP-box identification, siRNA with drug sensitivity

    PMID:35784486

    Open questions at the time
    • Functional consequence of the MLH1 interaction for MMR mechanistically thin
    • Single lab
  11. 2024 High

    Provided the structural basis for SHPRH-nucleosome recognition, showing acidic-patch engagement and a non-canonical SF2 ATPase contact with nucleosomal DNA.

    Evidence Cryo-EM structure at 2.8 Å with AlphaFold-Multimer prediction

    PMID:38979307

    Open questions at the time
    • Does not capture a SHPRH-PCNA or SHPRH-E2 complex
    • Catalytic cycle on a true substrate not visualized
  12. 2026 High

    Established the direct biochemical mechanism of PCNA modification, showing SHPRH ubiquitinates unmodified PCNA at Lys164 via the Ube2D family in a HIRAN-dependent manner on free and DNA-bound PCNA.

    Evidence Reconstituted in vitro ubiquitylation, MS confirmation of Lys164, HIRAN domain mutagenesis/deletion

    PMID:41990034

    Open questions at the time
    • In vivo contribution of Ube2D versus Ubc13-Mms2 not quantified
    • Regulation of HIRAN-PCNA engagement at forks not addressed

Open questions

Synthesis pass · forward-looking unresolved questions
  • The integration of SHPRH's replication-fork, nucleolar transcription, and non-PCNA substrate (FYN, MLH1) activities into a single regulatory logic remains unresolved.
  • No unified model linking nucleosome-ATPase activity to PCNA versus rDNA functions
  • Physiological nucleosomal ubiquitination substrates unidentified
  • Determinants choosing Ube2D versus Ubc13-Mms2 E2 usage in cells unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016874 ligase activity 4 GO:0140096 catalytic activity, acting on a protein 4 GO:0003677 DNA binding 1 GO:0042393 histone binding 1 GO:0140657 ATP-dependent activity 1
Localization
GO:0000228 nuclear chromosome 2 GO:0005634 nucleus 1 GO:0005730 nucleolus 1
Pathway
R-HSA-73894 DNA Repair 3 R-HSA-392499 Metabolism of proteins 2 R-HSA-4839726 Chromatin organization 2 R-HSA-69306 DNA Replication 2 R-HSA-74160 Gene expression (Transcription) 1
Partners
HLTFMLH1PCNARAD18TONEBPUBC13USP1

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2006 SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes and functions as a ubiquitin E3 ligase indispensable for Mms2-Ubc13-dependent K63-linked polyubiquitylation of PCNA, establishing it as a functional homolog of yeast Rad5 in error-free postreplicational repair. Co-immunoprecipitation, in vitro ubiquitylation assay, active-site analysis Proceedings of the National Academy of Sciences of the United States of America High 17108083
2006 SHPRH associates with PCNA, RAD18, and UBC13 and promotes MMS-induced PCNA polyubiquitination; shRNA-mediated reduction of SHPRH increases MMS sensitivity and enhances genomic instability, placing SHPRH in the Rad5-orthologous error-free bypass branch of postreplication repair. Co-immunoprecipitation, stable shRNA knockdown, genomic instability assays, MMS sensitivity assay The Journal of cell biology High 17130289
2008 HLTF, a second RAD5 homolog, interacts with UBC13, PCNA, and SHPRH and promotes K63-linked PCNA polyubiquitination cooperatively with SHPRH; reduction of either SHPRH or HLTF enhances spontaneous mutagenesis. Co-immunoprecipitation, siRNA knockdown, mutagenesis assay, MEF chromosome break analysis Proceedings of the National Academy of Sciences of the United States of America High 18719106
2011 SHPRH and HLTF act in a damage-specific manner: MMS promotes HLTF degradation and enables SHPRH interactions with Rad18 and polymerase κ for error-free bypass, while UV activates HLTF-dependent TLS and HLTF inhibits SHPRH function. This demonstrates coordinated regulation of the two main PRR branches by these RAD5 homologs. Co-immunoprecipitation, siRNA knockdown, mutagenesis assay, TLS polymerase recruitment assay Molecular cell High 21396873
2011 In Shprh/Hltf double-mutant mouse embryonic fibroblasts, PCNA polyubiquitination (PCNA-Ubn) is not abolished, demonstrating that an alternative E3 ligase exists for PCNA polyubiquitination beyond SHPRH and HLTF. Double-knockout mouse genetics, immunoblot for PCNA ubiquitination, B-cell somatic hypermutation assay DNA repair High 21269891
2013 The NMR solution structure of the SHPRH PHD domain adopts a canonical PHD-finger fold with a central two-stranded anti-parallel β-sheet flanked by interleaved zinc-binding sites; NMR titration experiments reveal that the SHPRH PHD domain does NOT specifically interact with H3-derived peptides regardless of K4 methylation status, suggesting an alternative function for this domain. Solution NMR structure determination, NMR titration experiments with histone H3 peptides Journal of biomolecular NMR High 23907177
2017 SHPRH localizes to ribosomal DNA (rDNA) promoters in nucleoli and facilitates rRNA transcription; the PHD domain mediates interaction with histone H3 when H3K4 is not trimethylated. SHPRH enrichment at the rDNA promoter requires CHD4, is inhibited by rapamycin (mTOR inhibition), starvation, or actinomycin D, and SHPRH physically interacts with the RNA Polymerase I complex. ChIP (chromatin immunoprecipitation), co-immunoprecipitation with RNA Pol I, shRNA knockdown, pharmacological inhibition (rapamycin, actinomycin D), subcellular fractionation/localization Proceedings of the National Academy of Sciences of the United States of America High 28400511
2019 SHPRH is a nucleosome-stimulated ATPase that binds equally well to dsDNA and nucleosome core particles but shows preference for nucleosomes with extranucleosomal DNA; nucleosomes (but not dsDNA) strongly stimulate SHPRH ATPase activity without conventional remodeling. SHPRH also functions as a potent nucleosome E3-ubiquitin ligase active with at least 7 different E2 enzymes; with UBE2D1, it catalyzes branched polyubiquitin linkages and linkages associated with DNA repair factor recruitment or proteasomal degradation. In vitro ATPase assay, nucleosome binding assay, E2 enzyme screen (26 E2s), in vitro ubiquitylation assay, mass spectrometry of ubiquitin linkages Epigenetics & chromatin High 31434570
2019 TonEBP (tonicity-responsive enhancer-binding protein) is recruited to DNA damage sites with bulky adducts and sequentially recruits E3 ubiquitin ligase SHPRH and then deubiquitinase USP1 to DNA damage sites, regulating the dynamics of PCNA polyubiquitination; the Rel-homology domain of TonEBP is essential for interaction with SHPRH and USP1. Co-immunoprecipitation, laser micro-irradiation with live-cell imaging of recruitment, siRNA knockdown, immunoblot for PCNA ubiquitination iScience Medium 31376680
2020 SHPRH contains a functional APIM (PCNA-interacting) motif, and nuclear localization of SHPRH depends on direct interaction with PCNA through this motif; mutation of APIM alters mutation spectra. APIM mutagenesis, nuclear localization assay, mutation spectrum analysis (SupF assay), overexpression International journal of molecular sciences Medium 31973093
2020 SHPRH knockout cells show reduced activation of checkpoint kinase CHK2 and MCM2 after MMS treatment, identifying SHPRH as a regulator of the CHK2-dependent DNA damage response branch. CRISPR/gene knockout, immunoblot for CHK2 and MCM2 phosphorylation, drug sensitivity assays Biomolecules Medium 32192191
2022 SHPRH interacts with MMR protein MLH1 (but not MSH2); this interaction is conserved from yeast Rad5, where a MIP-box motif mediates Mlh1 interaction. Depletion of SHPRH results in mild resistance to alkylating agents, suggesting a role in MMR-associated apoptotic response. Co-immunoprecipitation, yeast two-hybrid, bioinformatic MIP-box identification, siRNA knockdown with drug sensitivity assay Frontiers in cell and developmental biology Medium 35784486
2021 KMT2B (lysine methyltransferase 2B) elevates SHPRH expression via H3K4me3 modification at the SHPRH promoter; SHPRH in turn modulates FYN ubiquitination, promoting FYN protein degradation. ChIP for H3K4me3 at SHPRH promoter, co-immunoprecipitation, ubiquitination assay, overexpression/knockdown with immunoblot Cellular signalling Medium 34606908
2024 Cryo-EM structure of SHPRH bound to the nucleosome at 2.8 Å resolution confirms that SHPRH interacts with the H2A-H2B acidic patch of the nucleosome, reveals that the SHPRH ATPase engages a different nucleosomal DNA location than other SF2-type ATPases, and clarifies the structural roles of SHPRH domains in nucleosome recognition. Cryo-EM structure determination at 2.8 Å, AlphaFold-Multimer prediction bioRxiv (preprint) / also published as PMID:38979307preprint High 38979307
2026 SHPRH directly ubiquitinates unmodified PCNA at Lys164 using the Ube2D family of E2-conjugating enzymes (not only Ubc13-Mms2), acting robustly on both free and DNA-bound PCNA; this activity requires the SHPRH HIRAN domain for PCNA interaction. In vitro ubiquitylation assay with purified components, mass spectrometry to confirm Lys164 modification, HIRAN domain mutagenesis/deletion PloS one High 41990034
2018 SHPRH-146aa, a protein encoded by circ-SHPRH via IRES-driven translation, protects full-length SHPRH from ubiquitin-proteasome degradation, thereby stabilizing SHPRH and allowing SHPRH to function as an E3 ligase ubiquitinating PCNA to suppress glioblastoma cell proliferation. Overexpression/knockdown in glioblastoma cell lines, in vivo xenograft, co-immunoprecipitation, ubiquitin proteasome degradation assay, PCNA ubiquitination immunoblot Oncogene Medium 29343848

Source papers

Stage 0 corpus · 29 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2018 A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis. Oncogene 601 29343848
2008 Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks. Proceedings of the National Academy of Sciences of the United States of America 238 18719106
2006 Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Proceedings of the National Academy of Sciences of the United States of America 200 17108083
2006 Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination. The Journal of cell biology 168 17130289
2010 Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance. DNA repair 154 20096653
2011 SHPRH and HLTF act in a damage-specific manner to coordinate different forms of postreplication repair and prevent mutagenesis. Molecular cell 152 21396873
2018 Novel tumour suppressive protein encoded by circular RNA, circ-SHPRH, in glioblastomas. Oncogene 92 29706655
2011 HLTF and SHPRH are not essential for PCNA polyubiquitination, survival and somatic hypermutation: existence of an alternative E3 ligase. DNA repair 54 21269891
2003 Cloning and characterization of a novel gene, SHPRH, encoding a conserved putative protein with SNF2/helicase and PHD-finger domains from the 6q24 region. Genomics 37 12837266
2021 Circ-SHPRH suppresses cadmium-induced transformation of human bronchial epithelial cells by regulating QKI expression via miR-224-5p. Ecotoxicology and environmental safety 30 34082244
2017 SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner. Proceedings of the National Academy of Sciences of the United States of America 25 28400511
2019 The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase. Epigenetics & chromatin 21 31434570
2020 Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA). International journal of molecular sciences 20 31973093
2019 TonEBP Regulates PCNA Polyubiquitination in Response to DNA Damage through Interaction with SHPRH and USP1. iScience 16 31376680
2021 The Emerging Role of Circ-SHPRH in Cancer. OncoTargets and therapy 15 34285509
2021 Structural Characterization of the Hidden Peptide SHPRH-146aa Encoded by Non-Coding circ-SHPRH to Act as Tumor Suppressor. Applied biochemistry and biotechnology 12 33559759
2024 Therapeutic SHPRH-146aa encoded by circ-SHPRH dynamically upregulates P21 to inhibit CDKs in neuroblastoma. Cancer letters 11 39002691
2020 The Human RAD5 Homologs, HLTF and SHPRH, Have Separate Functions in DNA Damage Tolerance Dependent on The DNA Lesion Type. Biomolecules 11 32192191
2014 Analysis of SHPRH functions in DNA repair and immunoglobulin diversification. DNA repair 11 25311267
2011 DNA damage discrimination at stalled replication forks by the Rad5 homologs HLTF and SHPRH. Molecular cell 10 21504827
2022 Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair. Frontiers in cell and developmental biology 9 35784486
2013 PHD domain from human SHPRH. Journal of biomolecular NMR 9 23907177
2024 A peptide encoded by the circular form of the SHPRH gene induces apoptosis in neuroblastoma cells. PeerJ 6 38282862
2023 Circ-SHPRH in human cancers: a systematic review and meta-analysis. Frontiers in cell and developmental biology 6 37305675
2024 In silico screening identifies SHPRH as a novel nucleosome acidic patch interactor. bioRxiv : the preprint server for biology 5 38979307
2017 SHPRH as a new player in ribosomal RNA transcription and its potential role in homeostasis of ribosomal DNA repeats. Transcription 5 29139335
2021 KMT2B promotes SHPRH expression to regulate 131I sensitivity in thyroid carcinoma cells by affecting FYN protein stability. Cellular signalling 3 34606908
2026 SHPRH together with the Ube2D family of enzymes directly ubiquitinates PCNA at Lys164 in vitro. PloS one 0 41990034
2024 Integrative genomics identifies SHPRH as a tumor suppressor gene in lung adenocarcinoma that regulates DNA damage response. British journal of cancer 0 38890444

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