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- EMDB-29414: EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a ... -
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Basic information
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Title | EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 3 (open 9-1-1 and stably bound chamber DNA) | ||||||||||||
![]() | EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 3 (open 9-1-1 and stably bound chamber DNA) | ||||||||||||
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![]() | DNA damage repair / Rad24-RFC / 9-1-1 clamp / DNA clamp / alternative clamp loader / DNA damage signaling / DNA BINDING PROTEIN-DNA complex / CELL CYCLE-DNA complex | ||||||||||||
Function / homology | ![]() meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Rad17 RFC-like complex / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching ...meiotic DNA integrity checkpoint signaling / checkpoint clamp complex / DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Rad17 RFC-like complex / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / Translesion Synthesis by POLH / telomere maintenance via recombination / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / mitotic DNA replication checkpoint signaling / reciprocal meiotic recombination / sister chromatid cohesion / recombinational repair / mitotic intra-S DNA damage checkpoint signaling / mitotic sister chromatid cohesion / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / subtelomeric heterochromatin formation / meiotic cell cycle / mismatch repair / 3'-5' exonuclease activity / telomere maintenance / DNA damage checkpoint signaling / cellular response to ionizing radiation / nucleotide-excision repair / double-strand break repair via homologous recombination / DNA-templated DNA replication / double-strand break repair / double-stranded DNA binding / site of double-strand break / chromosome, telomeric region / damaged DNA binding / DNA repair / chromatin binding / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol Similarity search - Function | ||||||||||||
Biological species | synthetic construct (others) / ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.76 Å | ||||||||||||
![]() | Zheng F / Georgescu R / Yao YN / O'Donnell ME / Li H | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology. Authors: Fengwei Zheng / Roxana E Georgescu / Nina Y Yao / Michael E O'Donnell / Huilin Li / ![]() Abstract: Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the ...Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs. At a 10-nt gap we captured five Rad24-RFC-9-1-1 loading intermediates in which the 9-1-1 DNA entry gate varies from fully open to fully closed around DNA using ATPγS, supporting the emerging view that ATP hydrolysis is not needed for clamp opening/closing, but instead for dissociation of the loader from the clamp encircling DNA. The structure of Rad24-RFC-9-1-1 at a 5-nt gap shows a 180° axially rotated 3'-dsDNA which orients the template strand to bridge the 3'- and 5'- junctions with a minimum 5-nt ssDNA. The structures reveal a unique loop on Rad24 that limits the length of dsDNA in the inner chamber, and inability to melt DNA ends unlike RFC, thereby explaining Rad24-RFC's preference for a preexisting ssDNA gap and suggesting a direct role in gap repair in addition to its checkpoint role. | ||||||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 266.6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 28.9 KB 28.9 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 13.9 KB | Display | ![]() |
Images | ![]() | 75.6 KB | ||
Filedesc metadata | ![]() | 8.7 KB | ||
Others | ![]() ![]() | 262.1 MB 262.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.1 MB | Display | |
Data in XML | ![]() | 23 KB | Display | |
Data in CIF | ![]() | 29.7 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8fs5MC ![]() 8fs3C ![]() 8fs4C ![]() 8fs6C ![]() 8fs7C ![]() 8fs8C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 3 (open 9-1-1 and stably bound chamber DNA) | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.828 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: half map A
File | emd_29414_half_map_1.map | ||||||||||||
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Annotation | half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map B
File | emd_29414_half_map_2.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : Rad24-RFC-911 clamp-DNA
+Supramolecule #1: Rad24-RFC-911 clamp-DNA
+Supramolecule #2: DNA
+Supramolecule #3: Proteins complex
+Macromolecule #1: Checkpoint protein RAD24
+Macromolecule #2: Replication factor C subunit 4
+Macromolecule #3: Replication factor C subunit 3
+Macromolecule #4: Replication factor C subunit 2
+Macromolecule #5: Replication factor C subunit 5
+Macromolecule #6: DNA damage checkpoint control protein MEC3
+Macromolecule #7: DNA damage checkpoint control protein RAD17
+Macromolecule #8: DDC1 isoform 1
+Macromolecule #9: Template strand
+Macromolecule #10: Primer strand 1
+Macromolecule #11: Primer strand 2
+Macromolecule #12: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
+Macromolecule #13: MAGNESIUM ION
+Macromolecule #14: ADENOSINE-5'-DIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 64.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 1.3 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |