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- PDB-8tlt: Cryo-EM structure of Rev1(deltaN)-Polzeta-DNA-dCTP complex -

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Basic information

Entry
Database: PDB / ID: 8tlt
TitleCryo-EM structure of Rev1(deltaN)-Polzeta-DNA-dCTP complex
Components
  • (DNA polymerase delta ...) x 2
  • (DNA polymerase zeta ...) x 2
  • DNA (5'-D(*TP*AP*AP*TP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*GP*AP*AP*T)-3')
  • DNA (5'-D(P*CP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')
  • DNA repair protein REV1
KeywordsDNA BINDING PROTEIN/DNA / DNA repair / DNA replication / translesion DNA synthesis / DNA polymerase / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


delta DNA polymerase complex / H3-H4 histone complex chaperone activity / DNA amplification / deoxycytidyl transferase activity / zeta DNA polymerase complex / RNA-templated DNA biosynthetic process / DNA replication, removal of RNA primer / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI ...delta DNA polymerase complex / H3-H4 histone complex chaperone activity / DNA amplification / deoxycytidyl transferase activity / zeta DNA polymerase complex / RNA-templated DNA biosynthetic process / DNA replication, removal of RNA primer / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Termination of translesion DNA synthesis / lagging strand elongation / double-strand break repair via break-induced replication / DNA strand elongation involved in DNA replication / DNA metabolic process / error-free translesion synthesis / leading strand elongation / error-prone translesion synthesis / replication fork / nucleotide-excision repair / double-strand break repair via homologous recombination / base-excision repair / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / 4 iron, 4 sulfur cluster binding / DNA replication / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin / mitochondrion / DNA binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase zeta catalytic subunit / DNA polymerase delta/II small subunit family / DNA repair protein Rev1 / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / Mad2-like / HORMA domain ...DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase zeta catalytic subunit / DNA polymerase delta/II small subunit family / DNA repair protein Rev1 / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / Mad2-like / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / : / DNA polymerase-iota, thumb domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / BRCT domain, a BRCA1 C-terminus domain / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / DNA polymerase, Y-family, little finger domain / impB/mucB/samB family C-terminal domain / UmuC domain / DNA polymerase, Y-family, little finger domain superfamily / impB/mucB/samB family / UmuC domain profile. / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / POL31 isoform 1 / DNA repair protein REV1 / DNA polymerase zeta catalytic subunit / DNA polymerase zeta processivity subunit / DNA polymerase delta subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsMalik, R. / Aggarwal, A.K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R35-GM131780 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Cryo-EM structure of the Rev1-Polζ holocomplex reveals the mechanism of their cooperativity in translesion DNA synthesis.
Authors: Radhika Malik / Robert E Johnson / Iban Ubarretxena-Belandia / Louise Prakash / Satya Prakash / Aneel K Aggarwal /
Abstract: Rev1-Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the ...Rev1-Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the cryogenic electron microscopic structure of the Saccharomyces cerevisiae Rev1-Polζ holocomplex in the act of DNA synthesis (3.53 Å). We discovered that a composite N-helix-BRCT module in Rev1 is the keystone of Rev1-Polζ cooperativity, interacting directly with the DNA template-primer and with the Rev3 catalytic subunit of Polζ. The module is positioned akin to the polymerase-associated domain in Y-family TLS polymerases and is set ideally to interact with PCNA. We delineate the full extent of interactions that the carboxy-terminal domain of Rev1 makes with Polζ and identify potential new druggable sites to suppress chemoresistance from first-line chemotherapeutics. Collectively, our results provide fundamental new insights into the mechanism of cooperativity between Rev1 and Polζ in TLS.
History
DepositionJul 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 15, 2024Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Database references / Structure summary / Category: citation / citation_author / struct
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _struct.title
Revision 1.2Sep 25, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.3Oct 2, 2024Group: Data collection / Database references / Category: citation_author / em_admin
Item: _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase zeta catalytic subunit
D: DNA polymerase zeta processivity subunit
E: DNA polymerase zeta processivity subunit
F: DNA polymerase delta small subunit
G: DNA polymerase delta subunit 3
B: DNA repair protein REV1
P: DNA (5'-D(P*CP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')
T: DNA (5'-D(*TP*AP*AP*TP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*GP*AP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)453,46411
Polymers452,6058
Non-polymers8593
Water7,873437
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA polymerase zeta ... , 2 types, 3 molecules ADE

#1: Protein DNA polymerase zeta catalytic subunit / Protein reversionless 3


Mass: 177068.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: REV3, PSO1, YPL167C, P2535 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P14284, DNA-directed DNA polymerase
#2: Protein DNA polymerase zeta processivity subunit / Revertibility protein 7


Mass: 28791.654 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: REV7, YIL139C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38927

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DNA polymerase delta ... , 2 types, 2 molecules FG

#3: Protein DNA polymerase delta small subunit / SX2_G0007820.mRNA.1.CDS.1


Mass: 55987.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS3277, PACBIOSEQ_LOCUS3308 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PTG9
#4: Protein DNA polymerase delta subunit 3


Mass: 40377.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL32, YJR043C, J1626 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P47110

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Protein , 1 types, 1 molecules B

#5: Protein DNA repair protein REV1 / Reversionless protein 1


Mass: 112384.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: REV1, YOR346W, O6339 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P12689, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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DNA chain , 2 types, 2 molecules PT

#6: DNA chain DNA (5'-D(P*CP*CP*CP*TP*CP*CP*CP*CP*TP*AP*C)-3')


Mass: 3190.089 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (5'-D(*TP*AP*AP*TP*GP*GP*TP*AP*GP*GP*GP*GP*AP*GP*GP*GP*AP*AP*T)-3')


Mass: 6013.907 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 440 molecules

#8: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-DCP / 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE


Mass: 467.157 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O13P3
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 437 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1DNA complexCOMPLEX#1-#50MULTIPLE SOURCES
2DNA polymerase zeta catalytic subunit, DNA polymerase zeta processivity subunit, DNA polymerase delta small subunit, DNA polymerase delta subunit 3, DNA repair protein REV1COMPLEX#1-#41RECOMBINANT
3DNACOMPLEX#5-#61SYNTHETIC
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50.79 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 368486 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00419760
ELECTRON MICROSCOPYf_angle_d0.51926943
ELECTRON MICROSCOPYf_dihedral_angle_d9.1022822
ELECTRON MICROSCOPYf_chiral_restr0.043081
ELECTRON MICROSCOPYf_plane_restr0.0043292

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