Purification of an 86-70 kDa nuclear DNA-associated protein complex

Yaneva, Mariana; Ochs, Robert; McRorie, Donald K.; Zweig, Steve; Busch, Harris

doi:10.1016/0304-4165(85)90270-3

Cited by 75 publications

(58 citation statements)

References 10 publications

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“…The Ku80-EGFP fusion proteins were found in the nucleus of both HeLa-S3 and MCF-7 cells (Figures 3Ba and 4A), while EGFP alone was localized throughout the cell ( Figure 3Bb). This agreed well with the results obtained by immunocytochemical analysis (Reeves, 1985;Yaneva et al, 1985;Mimori et al, 1986;Koike et al, 1998). Since both Ku70 and Ku80 monomers are highly unstable compared to their heterodimers (Errami et al, 1996;Gu et al, 1997;Singleton et al, 1997), we were interested in studying the nuclear translocation of Ku80 and its heterodimer partner Ku70.…”

Section: Resultssupporting

confidence: 87%

“…Thus, we examined the cellular localization of the Ku70 and Ku80 proteins in HeLa-S3 cells using confocal laser scanning microscopy ( Figure 1A). Double staining using antibodies speci®c for the Ku70 and Ku80 subunits, respectively, resulted in uorescence mainly in the nucleoplasm of almost all the interphase cells screened, as reported in previous studies (Reeves, 1985;Yaneva et al, 1985;Mimori et al, 1990;Koike et al, 1998). However, in the course of our study, we found that the staining pattern of Ku80 did not completely coincide with that of Ku70 in some cells ( Figure 1A).…”

Section: Resultssupporting

confidence: 86%

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Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Koike¹,

Ikuta

Miyasaka³

et al. 1999

Oncogene

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Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation. Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations. Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku80 started during the late telophase/ early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70. Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70. To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and de®ned to a region spanning nine amino acid residues (positions 561 ± 569). The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these ®ndings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70.

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Section: Resultssupporting

confidence: 87%

Section: Resultssupporting

confidence: 86%

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Koike¹,

Ikuta

Miyasaka³

et al. 1999

Oncogene

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“…It consists of two polypeptides of 86 and 70 kDa (Yaneva et al, 1985). Ku is identical to a DNA-dependent ATPase isolated from HeLa cells (Cao et al, 1994) that had been previously reported to cofractionate with a 21S multiprotein complex competent for DNA synthesis from HeLa cells (Vishwanatha and Baril, 1990).…”

Section: Introductionmentioning

confidence: 90%

“…Ku antigen (autoantigen) is a heterodimeric (p70/p86) DNA-binding protein recognized by autoantibodies from the sera of certain patients with systemic rheumatic diseases (Mimori et al, 1981;Reeves, 1985;Yaneva et al, 1985;Mimori and Hardin, 1986). It consists of two polypeptides of 86 and 70 kDa (Yaneva et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

OBA/Ku86: DNA Binding Specificity and Involvement in Mammalian DNA Replication

Ruiz

Matheos

Price

et al. 1999

MBoC

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Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4 -OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of ϳ92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication. INTRODUCTIONKu antigen (autoantigen) is a heterodimeric (p70/p86) DNA-binding protein recognized by autoantibodies from the sera of certain patients with systemic rheumatic diseases (Mimori et al., 1981;Reeves, 1985;Yaneva et al., 1985;Mimori and Hardin, 1986). It consists of two polypeptides of 86 and 70 kDa (Yaneva et al., 1985). Ku is identical to a DNA-dependent ATPase isolated from HeLa cells (Cao et al., 1994) that had been previously reported to cofractionate with a 21S multiprotein complex competent for DNA synthesis from HeLa cells (Vishwanatha and Baril, 1990). Furthermore, the interaction of Ku antigen with a human DNA region (B48) containing a replication origin was reported (Tóth et al., 1993), and a novel ATP-dependent DNA unwinding enzyme, DNA helicase II (HDH II), was identified as Ku (Tuteja et al., 1994). Recently, Ochem et al. (1997) reported that the Ku70 subunit is the one associated with the helicase activity in the Ku70/Ku86 heterodimer. Moreover, a role for Ku70 as a tumor suppressor for murine T cell lymphoma has been suggested, because Ku70 deficiency facilitates neoplastic growth (Li et al., 1998). Ku has been shown to be the DNA-binding subunit of the DNA-dependent protein kinase (DNA-PK) holoenzyme Suwa et al., 1994), a nuclear component that phosphorylates a number of DNA-binding, regulatory proteins, including transcription factors (Sp1, p53), RNA polymerase II, topoisomerases I and II, Ku antigen, and SV-40 large T antigen (Anderson, 1993, and references therein). Although Ku has been characterized as a DNA end-binding protein, it was recently shown that it is also a sequence-specific DNA-binding protein, binding to negative regulatory element 1 (NRE1) in the long terminal repeat of...

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“…This protein is also referred to as p70/p80 (5) and the 86-70-kD protein complex (6,7). These antigens were identified on the basis oftheir reactivity with antibodies from patients with scleroderma-polymyositis overlap syndrome as well as with systemic lupus erythematosus and scleroderma.…”

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confidence: 99%

The autoantigen Ku is indistinguishable from NF IV, a protein forming multimeric protein-DNA complexes.

Stuiver

Coenjaerts

Vliet

1990

The Journal of Experimental Medicine

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We have isolated a cDNA encoding the 84-kD subunit of NFIV. Tryptic peptide sequences were identified within the coding sequences, confirming its proper identity. The primary sequence of the protein is identical to that of the large subunit of the Ku autoantigen. A missing NFIV peptide sequence was identified within the sequence of the small subunit of Ku. In addition, the proteins are identical in immunological aspects. We suggest that the Ku and NFIV proteins are identical. This connection adds new biochemical data to our knowledge of the Ku autoantigen.

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Purification of an 86-70 kDa nuclear DNA-associated protein complex

Cited by 75 publications

References 10 publications

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

OBA/Ku86: DNA Binding Specificity and Involvement in Mammalian DNA Replication

The autoantigen Ku is indistinguishable from NF IV, a protein forming multimeric protein-DNA complexes.

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