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 DNA-bound (HSA)kin17 protein 24 h after gamma 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.
Highlights
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]
After ␥ irradiation, replication protein A (RPA) concentrated in nuclear foci of very strong intensity and almost all the bright RPA foci co-localized with HSAkin17 (Fig. 6, B and C). These results suggested that HSAkin17 and RPA could cooperate in response to IR-induced DNA lesions
We assumed that (i) HSAkin17 and RPA belong to a same high molecular weight complex without direct interaction between them, (ii) only a small fraction of both proteins participate in the same nuclear foci, and (iii) constitutive amounts of both HSAkin17 and RPA proteins were too low to be detected under our experimental conditions
Summary
Cell Cultures—Human colorectal carcinoma RKO cells were obtained from M. Cloning of EBV vectors carrying HSAKIN17 cDNA in an antisense orientation has been performed as described elsewhere [23]. Transfected RKO cells carrying EBV plasmids were propagated in culture in the presence of 500 g/ml hygromycin B (Invitrogen). RKO clones transfected with the pB399as EBV vector carrying the HSAKIN17 cDNA-SV40 polyadenylation signal cartridge in an antisense orientation were termed RASK, for RKO antisense HSAKIN17 cDNA. Obtained after inoculation of recombinant His-tagged human HSAkin protein (His6-HSAkin17) purified by metal chelation and heparin column chromatography from baculovirus-infected Sf9 Spodoptera frugiperda cell extracts and injected in mice as described previously.. Purified immunoglobulin from rabbit polyclonal antibody anti-His6-HSAkin (IgG 77P) was obtained as described elsewhere.. 23 weeks after transfection, the cells were seeded as indicated in the presence of 500 g/ml hygromycin B.
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