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TitleStructures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology.
Journal, issue, pagesCell Rep, Vol. 42, Issue 7, Page 112694, Year 2023
Publish dateJul 25, 2023
AuthorsFengwei Zheng / Roxana E Georgescu / Nina Y Yao / Michael E O'Donnell / Huilin Li /
PubMed AbstractRad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. ...Rad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' end, thus presumably leaving 9-1-1 on duplex 3' ss/double-stranded DNA (dsDNA) after Rad24-RFC ejects from DNA. We captured five Rad24-RFC-9-1-1 loading intermediates using a 10-nt gap DNA. We also determined the structure of Rad24-RFC-9-1-1 using a 5-nt gap DNA. The structures reveal that Rad24-RFC is unable to melt DNA ends and that a Rad24 loop limits the dsDNA length in the chamber. These observations explain Rad24-RFC's preference for a preexisting gap of over 5-nt ssDNA and suggest a direct role of the 9-1-1 in gap repair with various TLS (trans-lesion synthesis) polymerases in addition to signaling the ATR kinase.
External linksCell Rep / PubMed:37392384 / PubMed Central
MethodsEM (single particle)
Resolution2.76 - 3.04 Å
Structure data

29412

8fs3

EMDB-29412: EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 1 (open 9-1-1 and shoulder bound DNA only)
PDB-8fs3: Structure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 1 (open 9-1-1 and shoulder bound DNA only)
Method: EM (single particle) / Resolution: 2.93 Å

29413

8fs4

EMDB-29413: EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 2 (open 9-1-1 ring and flexibly bound chamber DNA)
PDB-8fs4: Structure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 2 (open 9-1-1 ring and flexibly bound chamber DNA)
Method: EM (single particle) / Resolution: 2.94 Å

29414

8fs5

EMDB-29414: 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)
PDB-8fs5: Structure 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)
Method: EM (single particle) / Resolution: 2.76 Å

29415

8fs6

EMDB-29415: EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 4 (partially closed 9-1-1 and stably bound chamber DNA)
PDB-8fs6: Structure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 4 (partially closed 9-1-1 and stably bound chamber DNA)
Method: EM (single particle) / Resolution: 2.9 Å

29416

8fs7

EMDB-29416: EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 5 (closed 9-1-1 and stably bound chamber DNA)
PDB-8fs7: Structure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 10-nt gapped DNA in step 5 (closed 9-1-1 and stably bound chamber DNA)
Method: EM (single particle) / Resolution: 2.85 Å

29417

8fs8

EMDB-29417: EM map of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 5-nt gapped DNA (9-1-1 encircling fully bound DNA)
PDB-8fs8: Structure of S. cerevisiae Rad24-RFC loading the 9-1-1 clamp onto a 5-nt gapped DNA (9-1-1 encircling fully bound DNA)
Method: EM (single particle) / Resolution: 3.04 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

Source
  • synthetic construct (others)
  • saccharomyces cerevisiae (brewer's yeast)
KeywordsCELL CYCLE/DNA / 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

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