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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: 8.56). 08/2005; 24(29):4728-35. DOI: 10.1038/sj.onc.1208674
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ABSTRACT 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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    • "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; DOI:10.1111/mmi.12418 · 5.03 Impact Factor
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    • "RPA stimulates recombination reaction in vitro, especially when long DNA substrates are used (Park et al., 1996; Sugiyama and Kowalczykowski, 2002; Sung et al., 2003; Grimme et al., 2010). Furthermore, RPA-coated ssDNA has an essential role in the activation of DNA damage checkpoint (Zou and Elledge, 2003; Zou et al., 2003, 2006; Wu et al., 2005a; Choi et al., 2010; Liu et al., 2011). "
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    ABSTRACT: RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.
    Cell Biology International 05/2012; 36(8):713-20. DOI:10.1042/CBI20110633 · 1.64 Impact Factor
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    • "Several types of DNA damage result in the formation of regions of single stranded DNA (ssDNA) which is recognized by the single strand binding protein RPA [6] [7]. One of the first players recruited to sites of RPA-coated ssDNA is a PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 (9–1–1 complex) [8] [9]. Once loaded onto the chromatin, the 9–1–1 complex acts as a docking site for several enzymes involved in DNA damage signaling and repair [10] [11]. "
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    ABSTRACT: The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression.
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