Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

Majka, Jerzy; Chung, Brian Y.; Burgers, Peter M.

doi:10.1074/jbc.m400898200

Cited by 38 publications

(37 citation statements)

References 28 publications

(59 reference statements)

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“…3A, loading of PCNA by Elg1-RFC was also not observed (data not shown). This complex also failed to load the DNA damage checkpoint clamp Rad17/Mec3/Ddc1 onto primed DNA, under conditions that gave efficient loading by Rad24-RFC, leaving it currently as the only clamp loader with no specific biochemical function (data not shown) (29).…”

Section: Resultsmentioning

confidence: 99%

Replication Protein A-Directed Unloading of PCNA by the Ctf18 Cohesion Establishment Complex

Bylund

Burgers

2005

Molecular and Cellular Biology

122

145

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The replication clamp PCNA is loaded around DNA by replication factor C (RFC) and functions in DNA replication and repair. Regulated unloading of PCNA during the progression and termination of DNA replication may require additional factors. Here we show that a Saccharomyces cerevisiae complex required for the establishment of sister chromatid cohesion functions as an efficient unloader of PCNA. Unloading requires ATP hydrolysis. This seven-subunit Ctf18-RFC complex consists of the four small subunits of RFC, together with Ctf18, Dcc1, and Ctf8. Ctf18-RFC was also a weak loader of PCNA onto naked template-primer DNA. However, when the single-stranded DNA template was coated by the yeast single-stranded DNA binding protein replication protein A (RPA) but not by a mutant form of RPA or a heterologous single-stranded DNA binding protein, both binding of Ctf18-RFC to substrate DNA and loading of PCNA were strongly inhibited, and unloading predominated. Neither yeast RFC itself nor two other related clamp loaders, containing either Rad24 or Elg1, catalyzed significant unloading of PCNA. The Dcc1 and Ctf8 subunits of Ctf18-RFC, while required for establishing sister chromatid cohesion in vivo, did not function specifically in PCNA unloading in vitro, thereby separating the functionality of the Ctf18-RFC complex into two distinct paths.The process of sister chromatid cohesion ensures that replicated chromosomes are distributed equally to progeny cells during cell division. Cohesion is mediated through a large ring-like structure, cohesin, which is deposited on the chromosomes in the late G 1 phase of the cell cycle. Sister chromatid cohesion is established during S phase, presumably by the actual passage of the replication fork. Several models exist by which cohesin is proposed to interact with the duplicated chromosomes in order to mediate cohesion. These models are based either on the idea that one cohesin ring encircles both sister chromosomes or on the idea that cohesion is mediated by the interlocking of two cohesin rings, with each ring more peripherally associated with a daughter chromosome (reviewed in references 33, 34, and 48). If the first type of model is correct, the mere passage of the replication fork through a cohesin ring would invariably ensure that the two sister chromatids stay attached until mitosis. However, the disadvantage of this elegant solution to the chromosome sorting problem is that it may be problematic for the replication fork to pass through the estimated 30-to 40-nm hole of the cohesin ring. Recent studies have shown that cohesin is specifically redistributed along the chromosome to transcription termination sites in a transcription-dependent manner, suggesting that the actual transcription machinery may push the cohesin rings ahead of the transcription bubble (26). If this is caused by steric problems because of the size of the transcription apparatus, similar steric problems may also occur with passage of the replication fork. Failure to establish sister chromatid cohesion would resul...

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Section: Resultsmentioning

confidence: 99%

Replication Protein A-Directed Unloading of PCNA by the Ctf18 Cohesion Establishment Complex

Bylund

Burgers

2005

Molecular and Cellular Biology

122

145

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“…In vitro studies have shown that hRAD17-RFC as clamp loader recruits the 9-1-1 complex to the location of DSBs. 22,[32][33][34][35] Phosphorylation of hRAD17 by ATR upon DNA damage is necessary for the DNA damage checkpoint response. 31,36 Therefore, hRAD17 may be involved in DNA repair synthesis and mediate cell cycle regulation.…”

Section: Discussionmentioning

confidence: 99%

“…Loss of hRAD17 expression occurs frequently in HNSCC, is often due to genomic deletion, and may facilitate genomic instability in HNSCC. V V C 2007 Wiley Periodicals, Inc. Head Neck 30: [35][36][37][38][39][40][41][42]2008 Keywords: head and neck cancer; LOH; chromosomal instability; downregulation; hrad17 Cells respond to DNA damage by activating a highly coordinated DNA damage response. Failure to monitor and to signal DNA damage leads to genomic instability, which can ultimately lead to cancer formation due to dysregulation of critical genes that maintain cellular homeostasis.…”

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confidence: 99%

Downregulation of RAD17 in head and neck cancer

Zhao

Begum

et al. 2007

Head & Neck

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Background. DNA repair genes play a critical role in maintaining genome stability and have been implicated in tumorigenesis. Head and neck squamous cell carcinoma (HNSCC) often shows chromosomal instability. We examined the expression of human RAD17, a DNA damage cell cycle checkpoint gene, in primary head and neck cancer tissue.Methods. Significance analysis of microarrays was applied to expression array results examining more than 12,000 genes in 7 samples of primary HNSCC and 6 samples of normal control oral epithelial tissue. Additional confirmation was performed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in these samples and western blot with an additional 12 primary HNSCC and 7 normal samples, followed by loss of heterozygosity (LOH) analysis and quantitative PCR at the RAD17 locus.Results. Multiple checkpoint and DNA repair genes were downregulated in primary head and neck tumor tissue compared with normal control epithelial tissue, including hRAD17. Its Z-score and fold change were À2.5 and 0.39, respectively. The results of normalized, quantitative RT-PCR showed decreased expression of hRAD17 mRNA in tumor tissue (mean value 0.2166) when compared with normal tissue (mean value 0.3957, p < .05). Western blot demonstrated undetectable expression of hRAD17 protein in primary tumor tissue (0/12), while there was strong expression of hRAD17 protein in normal oral mucosal tissue (6/7). To determine possible mechanisms of inactivation, the hRAD17 locus at 5q13 was analyzed using microsatellite markers, showing 70% LOH in 30 primary HNSCCs. Quantitative PCR showed that RAD17 DNA copy number was decreased in the majority of head and neck tumor tissue samples.Conclusion. Loss of hRAD17 expression occurs frequently in HNSCC, is often due to genomic deletion, and may facilitate genomic instability in HNSCC.

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“…Proteins, Yeast Strains, and DNA-The checkpoint factors and DNA polymerase ␦ (pol ␦) were overproduced in yeast and purified as described previously (12,15,16). PCNA and RFC (a version lacking the N-terminal 172-amino-acid domain of Rfc1) were overproduced in Escherichia coli (17).…”

Section: Methodsmentioning

confidence: 99%

Replication Protein A Directs Loading of the DNA Damage Checkpoint Clamp to 5′-DNA Junctions

Majka

Binz

Wold

et al. 2006

Journal of Biological Chemistry

Self Cite

188

203

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The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial doublestranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPAmediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3-junction or a 5-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3-junctions was completely inhibited, thereby providing specificity to loading at 5-junctions. However, Rad17/3/1 loaded at 5-junctions can slide across double-stranded DNA to nearby 3-junctions and thereby affect the activity of proteins that act at 3-termini. These studies show a unique specificity of the checkpoint loader for 5-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.DNA damage elicits a broad range of cellular responses from DNA repair to inhibition of cell cycle progression. DNA damage checkpoints cause the arrest of cells at appropriate points in the cell cycle so that the integrity of the DNA can be restored (1). Although damage-induced checkpoints can be executed in many phases of the cell cycle, most of the progress in understanding the molecular details of this process has been made in the G 1 phase of the cell cycle, because its execution is unencumbered by complications related to the progression of DNA replication or chromosome segregation. In G 1 , for the DNA damage checkpoint to be executed in response to UV irradiation, repair of damage needs to be initiated by the nucleotide excision repair machinery (2). This finding suggests that gaps made during the progression of nucleotide excision repair are targets for the checkpoint machinery.Gaps in the double-stranded DNA contain three structural elements, a region of single-stranded DNA (ssDNA), 2 a ss-ds DNA junction with a 5Ј-terminus (5Ј-junction), and a ss-ds DNA junction with a 3Ј-terminus (3Ј-junction). Although the ssDNA is a target for binding by the single-stranded binding protein RPA, the 5Ј-and 3Ј-junction can be targeted by a variety of proteins, including nucleases (to either junction), DNA polymerases (to 3Ј-junctions), and circular clamp proteins. The replication clamp proliferating cell nuclear antigen (PCNA) is uniquely targeted to 3Ј-junctions and in that capacity serves as a processivity factor for several DNA polymerases and for a large number of other proteins that participate in various DNA metabolic pathways (reviewed in Ref.3). Much less is known about the PCNA-r...

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Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

Cited by 38 publications

References 28 publications

Replication Protein A-Directed Unloading of PCNA by the Ctf18 Cohesion Establishment Complex

Replication Protein A-Directed Unloading of PCNA by the Ctf18 Cohesion Establishment Complex

Downregulation of RAD17 in head and neck cancer

Replication Protein A Directs Loading of the DNA Damage Checkpoint Clamp to 5′-DNA Junctions

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