Biochemical Characterization of DNA Damage Checkpoint Complexes: Clamp Loader and Clamp Complexes with Specificity for 5′ Recessed DNA

Evolutionarily conserved shelterin complex is essential for telomere maintenance in the fission yeast Schizosaccharomyces pombe. Elimination of the fission yeast shelterin subunit Ccq1 causes progressive loss of telomeres due to the inability to recruit telomerase, activates the DNA damage checkpoint, and loses heterochromatin at telomere/subtelomere regions due to reduced recruitment of the heterochromatin regulator complex Snf2/histone deacetylase–containing repressor complex (SHREC). The shelterin subunit Tpz1TPP1 directly interacts with Ccq1 through conserved C-terminal residues in Tpz1TPP1, and tpz1 mutants that fail to interact with Ccq1 show telomere shortening, checkpoint activation, and loss of heterochromatin. While we have previously concluded that Ccq1-Tpz1TPP1 interaction contributes to Ccq1 accumulation and telomerase recruitment based on analysis of tpz1 mutants that fail to interact with Ccq1, another study reported that loss of Ccq1-Tpz1TPP1 interaction does not affect accumulation of Ccq1 or telomerase. Furthermore, it remained unclear whether loss of Ccq1-Tpz1TPP1 interaction affects SHREC accumulation at telomeres. To resolve these issues, we identified and characterized a series of ccq1 mutations that disrupt Ccq1-Tpz1TPP1 interaction. Characterization of these ccq1 mutants established that Ccq1-Tpz1TPP1 interaction contributes to optimal binding of the Ccq1-SHREC complex, and is critical for Rad3ATR/Tel1ATM-dependent Ccq1 Thr93 phosphorylation and telomerase recruitment.

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Rad24-RFC loads the 9-1-1 clamp by inserting DNA from the top of a wide-open ring, opposite the mechanism of RFC/PCNA

10.1101/2021.10.01.462756 ◽

2021 ◽

Author(s):

Fengwei Zheng ◽

Roxana E. Georgescu ◽

Nina Y. Yao ◽

Michael E. O’Donnell ◽

Huilin Li

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Atp Hydrolysis ◽

Dna Damage Checkpoint ◽

Clamp Loader ◽

Dna Binding Site ◽

Clamp Loading ◽

Open Ring ◽

Binding Residues ◽

Higher Eukaryotes

ABSTRACTIn response to DNA damage, the ring-shaped 9-1-1 clamp is loaded onto 5’ recessed DNA to arrest the cell cycle and activate the DNA damage checkpoint. The 9-1-1 clamp is a heterotrimeric ring that is loaded in S. cerevisiae by Rad24-RFC, an alternative clamp loader in which Rad24 replaces the Rfc1 subunit in the RFC1-5 clamp loader of PCNA. Unlike RFC that loads the PCNA ring onto a 3’-ss/ds DNA junction, Rad24-RFC loads the 9-1-1 ring onto a 5’-ss/ds DNA junction, a consequence of DNA damage. The underlying 9-1-1 clamp loading mechanism has been a mystery. Here we report two 3.2-Å cryo-EM structures of Rad24-RFC bound to DNA and either a closed or 27 Å open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. The Rad24 subunit specifically recognizes the 5’-DNA junction and holds ds DNA outside the clamp loader and above the plane of the 9-1-1 ring, rather than holding DNA inside and below the clamp as in RFC. The 3’ ssDNA overhang is required to obtain the structure, and thus confers a second DNA binding site. The bipartite DNA binding by Rad24-RFC suggests that ssDNA may be flipped into the open 9-1-1 ring, similar to ORC-Cdc6 that loads the Mcm2-7 ring on DNA. We propose that entry of ssDNA through the 9-1-1 ring triggers the ATP hydrolysis and release of the Rad24-RFC. The key DNA binding residues are conserved in higher eukaryotes, and thus the 9-1-1 clamp loading mechanism likely generalizes.

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Rfc4 Interacts with Rpa1 and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.21.11.3725-3737.2001 ◽

2001 ◽

Vol 21 (11) ◽

pp. 3725-3737 ◽

Cited By ~ 72

Author(s):

Hee-Sook Kim ◽

Steven J. Brill

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Essential Role ◽

Large Subunit ◽

Small Subunit ◽

Dna Damage Checkpoint ◽

Physical Interaction ◽

Clamp Loader ◽

Synthesis Inhibitor ◽

Factor C

ABSTRACT The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such asrfa1-t11, are lethal in combination withrfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint geneRAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G1/S DNA damage checkpoint response and that both therfc4-2 and rfa1-t11 strains are defective in the G2/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.

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Purification and characterization of human DNA damage checkpoint Rad complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.201373498 ◽

2001 ◽

Vol 98 (20) ◽

pp. 11236-11241 ◽

Cited By ~ 137

Author(s):

L. A. Lindsey-Boltz ◽

V. P. Bermudez ◽

J. Hurwitz ◽

A. Sancar

Keyword(s):

Dna Damage ◽

Dna Damage Checkpoint ◽

Purification And Characterization ◽

Human Dna

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Isolation and characterization of the Schizosaccharomyces pombe rhp9 gene: a gene required for the DNA damage checkpoint but not the replication checkpoint

Nucleic Acids Research ◽

10.1093/nar/25.11.2138 ◽

1997 ◽

Vol 25 (11) ◽

pp. 2138-2146 ◽

Cited By ~ 70

Author(s):

J Willson

Keyword(s):

Dna Damage ◽

Schizosaccharomyces Pombe ◽

Dna Damage Checkpoint ◽

Replication Checkpoint ◽

Isolation And Characterization

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Plant Pathology Diagnosed by X-Ray Microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129152 ◽

1987 ◽

Vol 45 ◽

pp. 974-975

Author(s):

J. H. Resau ◽

N. Howell ◽

S. H. Chang

Keyword(s):

Electron Microscopy ◽

Plant Pathology ◽

Biochemical Characterization ◽

Nutrient Deficiency ◽

Green Leaf ◽

Parasitic Infestation ◽

X Ray

Spinach grown in Texas developed “yellow spotting” on the peripheral portions of the leaves. The exact cause of the discoloration could not be determined as there was no evidence of viral or parasitic infestation of the plants and biochemical characterization of the plants did not indicate any significant differences between the yellow and green leaf portions of the spinach. The present study was undertaken using electron microscopy (EM) to determine if a micro-nutrient deficiency was the cause for the discoloration.Green leaf spinach was collected from the field and sent by express mail to the EM laboratory. The yellow and equivalent green portions of the leaves were isolated and dried in a Denton evaporator at 10-5 Torr for 24 hrs. The leaf specimens were then examined using a JEOL 100 CX analytical microscope. TEM specimens were prepared according to the methods of Trump et al.

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