Structure of the HLTF HIRAN domain and its functional implications in regression of a stalled replication fork

2020 ◽  
Vol 76 (8) ◽  
pp. 729-735
Author(s):  
Asami Hishiki ◽  
Mamoru Sato ◽  
Hiroshi Hashimoto
Keyword(s):  
Dna Binding ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
Structural Information ◽  
E3 Ubiquitin Ligase ◽  
Dna Helicase ◽  
Structural Basis ◽  
The Past ◽  
Functional Implications ◽  
Fork Regression

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog that is found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and is a pivotal protein in template-switched DNA synthesis that allows DNA replication to continue even in the presence of DNA damage by utilizing a newly synthesized undamaged strand as a template. In addition, HLTF has a DNA-binding domain termed HIRAN (HIP116 and RAD5 N-terminal). HIRAN has been hypothesized to play a role in DNA binding; however, the structural basis of its role in DNA binding has remained unclear. In the past five years, several crystal structures of HIRAN have been reported. These structures revealed new insights into the molecular mechanism underlying DNA binding by HIRAN. Here, the structural information on HIRAN is summarized and the function of HIRAN in recognizing the 3′-terminus of the daughter strand at a stalled replication fork and the implications for its involvement in fork regression are discussed.

scholarly journals Structural basis for the multi-activity factor Rad5 in replication stress tolerance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Miaomiao Shen ◽  
Nalini Dhingra ◽  
Quan Wang ◽  
Chen Cheng ◽  
Songbiao Zhu ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
Replication Stress ◽  
Spatial Arrangement ◽  
Structural Basis ◽  
Dna Lesion ◽  
Activity Factor ◽  
Fork Regression ◽  
New Type

AbstractThe yeast protein Rad5 and its orthologs in other eukaryotes promote replication stress tolerance and cell survival using their multiple activities, including ubiquitin ligase, replication fork remodeling and DNA lesion targeting activities. Here, we present the crystal structure of a nearly full-length Rad5 protein. The structure shows three distinct, but well-connected, domains required for Rad5’s activities. The spatial arrangement of these domains suggest that different domains can have autonomous activities but also undergo intrinsic coordination. Moreover, our structural, biochemical and cellular studies demonstrate that Rad5’s HIRAN domain mediates interactions with the DNA metabolism maestro factor PCNA and contributes to its poly-ubiquitination, binds to DNA and contributes to the Rad5-catalyzed replication fork regression, defining a new type of HIRAN domains with multiple activities. Our work provides a framework to understand how Rad5 integrates its various activities in replication stress tolerance.

scholarly journals Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12

Open Biology ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 200041 ◽  
Author(s):  
Zhuoyao Chen ◽  
Gregory A. Wasney ◽  
Sarah Picaud ◽  
Panagis Filippakopoulos ◽  
Masoud Vedadi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
E3 Ligases ◽  
Kelch Domain ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
New Protein

Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.

scholarly journals Mms1 and Mms22 stabilize the replisome during replication stress

2011 ◽  
Vol 22 (13) ◽  
pp. 2396-2408 ◽  
Author(s):  
Jessica A. Vaisica ◽  
Anastasija Baryshnikova ◽  
Michael Costanzo ◽  
Charles Boone ◽  
Grant W. Brown
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
E3 Ubiquitin Ligase ◽  
Replication Stress ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Binding Partners ◽  
Efficient Recovery ◽  
Stalled Replication Forks

Mms1 and Mms22 form a Cul4Ddb1-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101Mms1/Mms22ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1—Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.

scholarly journals Structural Basis of the Activation and Degradation Mechanisms of the E3 Ubiquitin Ligase Nedd4L

Structure ◽  
2014 ◽  
Vol 22 (10) ◽  
pp. 1446-1457 ◽  
Author(s):  
Albert Escobedo ◽  
Tiago Gomes ◽  
Eric Aragón ◽  
Pau Martín-Malpartida ◽  
Lidia Ruiz ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
Degradation Mechanisms

scholarly journals Maturation of telomere 3’-overhangs is linked to the replication stress response

2020 ◽  
Author(s):  
Erin E. Henninger ◽  
Pascale Jolivet ◽  
Emilie Fallet ◽  
Mohcen Benmounah ◽  
Zhou Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Stress Response ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
Damage Tolerance ◽  
Dna Helicase ◽  
Dna Damage Tolerance ◽  
Telomeric Repeats ◽  
Telomere Replication

AbstractPassage of the replication fork through telomeric repeats necessitates additional DNA processing by DNA repair factors, to regenerate the terminal 3’-overhang structure at leading telomeres. These factors are prevented from promoting telomeric recombination or fusion by an uncharacterized mechanism. Here we show that Rad5, a DNA helicase and ubiquitin ligase involved in the DNA damage tolerance pathway, participates in this mechanism. Rad5 is enriched at telomeres during telomere replication. Accelerated senescence seen in the absence of telomerase and Rad5, can be compensated for by a pathway involving the Rad51 recombinase and counteracted by the helicase Srs2. However, this pathway is only active at short telomeres. Instead, the ubiquitous activity of Rad5 during telomere replication is necessary for the proper reconstitution of the telomeric 3’-overhang, indicating that Rad5 is required to coordinate telomere maturation during telomere replication.

scholarly journals Structural basis for feedforward control in the PINK1/parkin pathway

2021 ◽  
Author(s):  
Véronique Sauvé ◽  
George Sung ◽  
Emma MacDougall ◽  
Guennadi Kozlov ◽  
Anshu Saran ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Feedforward Control ◽  
E3 Ubiquitin Ligase ◽  
Evolutionary Development ◽  
Structural Basis ◽  
Intermediate Step ◽  
Vinyl Sulfone ◽  
Mitochondrial Quality Control ◽  
High Affinity Site ◽  
Nmr Titrations

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observe that phospho-ubiquitin can bind to two distinct sites on parkin, a high affinity site on RING1 that controls parkin localization, and a low affinity site on RING0 that releases parkin autoinhibition. Surprisingly, NMR titrations and ubiquitin vinyl sulfone assays show that the RING0 site has higher affinity for phospho-ubiquitin than the phosphorylated Ubl. Parkin could be activated by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic a mitochondrion bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.

scholarly journals Parvovirus Initiator Protein NS1 and RPA Coordinate Replication Fork Progression in a Reconstituted DNA Replication System

2002 ◽  
Vol 76 (13) ◽  
pp. 6518-6531 ◽  
Author(s):  
Jesper Christensen ◽  
Peter Tattersall
Keyword(s):  
Dna Binding ◽  
Replication Fork ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Small Subunit ◽  
Dna Helicase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Initiator Protein

ABSTRACT We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5′ end and displacing the other strand while progressing in a 3′-to-5′ direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5′ end of the nick in the process and providing a 3′ OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO4 oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase δ, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

Structural basis of K63-ubiquitin chain formation by the Gordon-Holmes syndrome RBR E3 ubiquitin ligase RNF216

2022 ◽  
Author(s):  
Thomas R. Cotton ◽  
Simon A. Cobbold ◽  
Jonathan P. Bernardini ◽  
Lachlan W. Richardson ◽  
Xiangyi S. Wang ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
Ubiquitin Chain ◽  
Chain Formation

scholarly journals Structural analysis of the LDL receptor–interacting FERM domain in the E3 ubiquitin ligase IDOL reveals an obscured substrate-binding site

2020 ◽  
Vol 295 (39) ◽  
pp. 13570-13583
Author(s):  
Luca Martinelli ◽  
Athanassios Adamopoulos ◽  
Patrik Johansson ◽  
Paul T. Wan ◽  
Jenny Gunnarsson ◽  
...  
Keyword(s):  
Binding Site ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Full Length ◽  
Scattering Data ◽  
Lipid Lowering ◽  
Structural Basis ◽  
Ferm Domain

Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.

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