Ionizing Radiation Triggers Chromatin-bound kin17 Complex Formation in Human Cells

Abstract

The human DNA-binding HSA kin17 protein cross-reacts with antibodies raised against the stress-activated Escherichia coli RecA protein. We show here that HSA kin17 protein is directly associated with chromosomal DNA as judged by cross-linking experiments on living cells. We detected increased amounts of DNAbound HSA kin17 protein 24 h after ␥ irradiation, with 2.6-fold more HSA kin17 molecules after 6 Gy of irradiation (46,000 -117,000 molecules). At this time we observed that highly proliferating RKO cells displayed the concentration and co-localization of HSA kin17 and replication protein A in nucleoplasmic foci. Our results suggest that 24 h post-irradiation HSA kin17 protein may localize at the sites of unrepaired DNA damages. RKO clones expressing an HSA KIN17 antisense transcript (RASK.5 and RASK.13 cells) revealed that reduced HSA kin17 protein levels are correlated with a decrease in clonogenic cell growth and cell proliferation, as well as an accumulation of cells in early and mid-S phase. Taken together our observations support the idea that HSA kin17 protein is a DNA maintenance protein involved in the cellular response to the presence of DNA damage and suggest that it helps to overcome the perturbation of DNA replication produced by unrepaired lesions. Ionizing radiation (IR)1 induces a large range of DNA damage, including DNA double-strand breaks (DSBs), which represent a major threat to the integrity of mammalian genomes through chromosomal breakages and rearrangements (1). In mammalian cells, DSBs are repaired either by the homologous recombinational repair or by nonhomologous end joining (2, 3). DSB repair pathways are usually characterized by the sequestration of many factors into discrete nuclear foci at the sites of DNA lesions and until completion of DSB repair (4). Some of the proteins belonging to these pathways act as a sensor for DNA damage or are involved in cell cycle checkpoints. This is the case for the histone H2AX, 53BP1, RPA, Rad51, BRCA1, or the Mre11-Rad50-Nbs1 nuclease complex (4 -8). For instance, the tumor suppressor gene BRCA1, previously involved in the regulation of the replication checkpoint and transcription-coupled repair, forms a multiprotein complex with Mre11-Rad50-Nbs1 and other proteins following irradiation, termed as BASC (BRCA1-associated surveillance complex), which may serve as a sensor of DNA lesions (8,9).In this cascade of IR-induced proteins forming nuclear foci, we characterized here the HSA kin17 protein. Murine MMU kin17 protein was identified on the basis of a cross-reactivity with antibodies raised against the Escherichia coli RecA protein, a key enzyme in homologous recombination and recombinational repair of damaged DNA (10, 11). This cross-reactivity stemmed from a sequence homology stretching over 39 amino acids highly conserved during evolution (12). This domain is located in the carboxyl-terminal region of the E. coli RecA protein, a region involved in the regulation of DNA binding (13). Recent data show that kin17 proteins are highly conserved d...

KIN17 encodes an RNA-binding protein and is expressed during mouse spermatogenesis

“…Biochemical approaches indicated that this is the case, giving further support to the previously reported data. Indeed, mouse KIN17 protein binds to DNA in vitro (Angulo et al, 1991;Biard et al, 2002;Mazin et al, 1994b) and in HeLa cells and is associated with the nuclear matrix in cultured human cells . Most germ cells express KIN17.…”

Development of New EBV-Based Vectors for Stable Expression of Small Interfering RNA to Mimick Human Syndromes: Application to NER Gene Silencing

“…Procedures are described elsewhere (38). We used purified immunoglobulin G K36 and immunoglobulin K58 directed against HSA kin17 protein; monoclonal antibody anti-Ku70 (clone N3H10, Lab Vision/Neomarkers, Fremont, CA); antiKu80 (clone 111, Vision/Neomarkers), anti-RPA70 (clone RPA70-9, Oncogene Research Products, Calbiochem, Darmstadt, Germany), a-tubulin (clone B-5-1-2, Sigma Chemical Co., St. Louis, MO), proliferating cell nuclear antigen (clone PC10, Novo Castra, Newcastle, United Kingdom), p34cdc2 (clone sc54, Santa Cruz Biotechnology, Santa Cruz Biotechnology Inc., Santa Cruz, CA), pAbaXPC (a gift from Wim Vermeulen), and monoclonal antibody XPA (a gift from Yue Zou).…”

The Human Stress-Activated Protein kin17 Belongs to the Multiprotein DNA Replication Complex and Associates In Vivo with Mammalian Replication Origins

“…Indeed, kin17 forms intranuclear foci and accumulates in the nuclei of proliferating cells (32). Strikingly, kin17 concentrated in large nuclear foci associated with RPA after gamma irradiation (7). Cells presenting low levels of this protein also showed a prolongation of the S phase of the cell cycle associated with an accumulation of cells in early and mid-S phase, a decreased rate of DNA synthesis, and an increased sensitivity to gamma irradiation (7,17).…”

“…Strikingly, kin17 concentrated in large nuclear foci associated with RPA after gamma irradiation (7). Cells presenting low levels of this protein also showed a prolongation of the S phase of the cell cycle associated with an accumulation of cells in early and mid-S phase, a decreased rate of DNA synthesis, and an increased sensitivity to gamma irradiation (7,17). Besides, we have reported a physical interaction between human kin17 and simian virus 40 (SV40) large T antigen leading to both in vitro and in vivo DNA synthesis inhibition (30,47).…”

“…kin17 is a 45-kDa nuclear protein conserved during evolution, ubiquitously expressed in mammals (31). The main features of kin17 are its abilities to (i) bind directly to chromosomal DNA in human cells (7) and to RNA in mouse germ cells (56), (ii) bind preferentially to curved DNA found at the hot spots of illegitimate recombination (45, 46), (iii) complement the functions of a bacterial nucleoid protein called H-NS which binds to curved DNA and controls gene expression (66), and (iv) be upregulated after UV and ionizing radiations (6,7,9,32,42). Recently, a large-scale proteomic study of the human spliceosome-associated factors identified kin17 protein among 96 novel proteins related to splicing/mRNA processing, transcription, and cell cycle regulation (57).…”