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

Abstract

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G 1 /S border and throughout the S phase and was negligible in both G 0 and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase ␣. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an ϳ600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.The kin17 protein was initially identified based on the crossreacting property of antibodies raised against the stress-activated Escherichia coli RecA protein. kin17 displays a common epitope with the RecA protein and shares 47% homology over a 40-residue stretch in the RecA C-terminal region (2). In RecA protein, this region is involved in the regulation of DNA binding and in the SOS response (33). 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). A link between the ...

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“…Established KIN17 KD HeLa cells exhibited moderate sensitivity to UVC with about 40% survival after 4 J/m 2 , in comparison with 84% survival of control cells. These data are consistent with the previously reported implication of HSA kin17 protein in nucleic acid metabolism like replication and RNA processing (18)(19)(20)(21).…”

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“…Consequently, the discrete distribution of bent DNA within nuclei would precede the above-described pattern. Indeed, ultrastructural assays in cultured human cells colocalized the human KIN17 with newly synthesized DNA labeled with bromodeoxyuridine as well as with DNA replication-associated markers, such as the proliferating cell nuclear antigen, the replication protein A, or the DNA polymerase-a (Biard et al, 2003;Miccoli et al, 2005). These observations point out that KIN17 is associated with the nonchromatin nuclear structures named replication foci.…”

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