Interaction and colocalization of Rad9/Rad1/Hus1 checkpoint complex with replication protein A in human cells

Department of Biochemistry and Molecular Biology, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA.
Oncogene (Impact Factor: 8.46). 08/2005; 24(29):4728-35. DOI: 10.1038/sj.onc.1208674
Source: PubMed


Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-, 32-, and 14-kDa (RPA70, RPA32, RPA14, respectively). It is a protein essential for most cellular DNA metabolic pathways. Checkpoint proteins Rad9, Rad1, and Hus1 form a clamp-like complex which plays a central role in the DNA damage-induced checkpoint response. In this report, we presented the evidence that Rad9-Rad1-Hus1 (9-1-1) complex directly interacted with RPA in human cells, and this interaction was mediated by the binding of Rad9 protein to both RPA70 and RPA32 subunits. In addition, the cellular interaction of 9-1-1 with RPA or hyperphosphorylated RPA was stimulated by UV irradiation or camptothecin treatment in a dose-dependent manner. Such treatments also resulted in the colocalization of the nuclear foci formed with the two complexes. Consistently, knockdown of the RPA expression in cells by the small interference RNA (siRNA) blocked the DNA damage-dependent chromatin association of 9-1-1, and also inhibited the 9-1-1 complex formation. Taken together, our results suggest that 9-1-1 and RPA complexes collaboratively function in DNA damage responses, and that the RPA may serve as a regulator for the activity of 9-1-1 complex in the cellular checkpoint network.

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    • "These gaps in the genome are detected by the sequence-independent ssDNA-binding Replication Protein A (RPA) complex in conjunction with the 9-1-1 complex (Rad9-Hus1-Rad1). The recruitment of these complexes to a singlestranded gap results in the activation of the ATR kinase, formation of a DNA damage foci, and inhibition of cell cycle progression [15] [16]. The higher affinity of the POT1/TPP1 dimer for telomeric 3 0 overhangs is sufficient to block the recognition of these sites by the more abundant RPA and 9-1-1 complexes. "
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    ABSTRACT: The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5'-TTAGGGTTAG-3' and forms a dimer with the TPP1 protein. The dimer is recruited to telomeres, either directly or as part of the Shelterin complex. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5'-TTAGGG whereas OB2 binds to the downstream TTAG-3'. Studies of POT1 proteins from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected. To explore this possibility in humans, we have used SELEX to reexamine the sequence-specificity of the protein. Using human POT1 as a selection matrix, high-affinity DNA ligands were selected from a pool of randomized single-stranded oligonucleotides. After six successive rounds of selection, two classes of high-affinity targets were obtained. The first class was composed of oligonucleotides containing a cognate POT1 binding sites (5'-TTAGGGTTAG-3'). The second and more abundant class was made of molecules that carried a novel non-telomeric consensus: 5'-TNCANNAGKKKTTAGG-3' (where K=G/T and N=any base). Binding studies showed that these non-telomeric sites were made of an OB1-binding motif (TTAGG) and a non-telomeric motif (NT motif), with the two motifs recognized by distinct regions of the OB1 domain. POT1 interacted with these non-telomeric binding sites with high affinity and specificity, even when bound to its dimerization partner TPP1. This intrinsic ability of POT1 to recognize NT motifs raises the possibility that the protein may fulfill additional functions at certain non-telomeric locations of the genome, in perhaps gene transcription, replication, or repair. Copyright © 2015. Published by Elsevier B.V.
    Biochimie 04/2015; 115. DOI:10.1016/j.biochi.2015.04.015 · 2.96 Impact Factor
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    • "This was carried out to ensure a uniform cell cycle distribution upon subsequent exposure to IR. Cells were then exposed to 10Gy IR and harvested at progressive time-points in the presence or absence of DNase I, which serves to liberate chromatin-bound proteins that might otherwise precipitate during cell lysis [44–47]. As shown in the third panel of Figure 3, TLK1 and Rad9 were found to interact constitutively. "
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    ABSTRACT: Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.
    PLoS ONE 12/2013; 8(12):e85859. DOI:10.1371/journal.pone.0085859 · 3.23 Impact Factor
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    • "Despite our efforts, testing different cell lysis protocols and DNA damaging conditions, we did not witness a stable LmHus1/LmRpa1 interaction (Fig. S5B). However, confocal IF analysis, presented at the last column of Fig. 4C, showed that signals corresponding to the LmHus1 and LmRpa1 staining are partially overlapping, indicating the colocalization between these two proteins as in other eukaryotic cells (Wu et al., 2005). We did not detect significant change in the proportion of LmHus1 and LmRpa1 colocalization upon genotoxic stress. "
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    ABSTRACT: Genotoxic stress activates checkpoint-signaling pathways leading to cell cycle arrest and DNA repair. In many eukaryotes, the Rad9-Hus1-Rad1 (9-1-1) checkpoint complex participates in the early steps of the DNA damage response to replicative stress and is a pivotal contributor to genome homeostasis. The remarkable genome plasticity of the protozoan Leishmania hints at a peculiar DNA metabolism in these ancient eukaryotes. Therefore, we set out to investigate the existence of homologues of the 9-1-1 components in Leishmania major and found that LmHus1 and LmRad9 are phylogenetically related to the 9-1-1 complex subunits from other eukaryotes. Altered levels of LmHus1 and LmRad9 affected the parasite ability to manage genotoxic stress and LmHus1-defficent cells were defective in controlling cell cycle progression in response to genotoxic stress. Upon DNA damage, LmHus1 was recruited to the chromatin and co-localized with the single stranded DNA binding protein LmRpa1. Also, LmHus1 interacted with LmRad9 to form a DNA damage responsive complex in vivo. Altogether, our data strongly indicate the participation of LmHus1, LmRad9 and LmRpa1 in the L. major DNA damage response and suggest their involvement in genome maintenance mechanisms.
    Molecular Microbiology 10/2013; 90(5). DOI:10.1111/mmi.12418 · 4.42 Impact Factor
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