Sequence-specific Binding of Ku Autoantigen to Single-stranded DNA

Torrance, Heather; Giffin, Ward; Rodda, David J.; Pope, L. S.; Haché, Robert J.G.

doi:10.1074/jbc.273.33.20810

Cited by 22 publications

(24 citation statements)

References 88 publications

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“…Recently, the sequence-specific binding of Ku autoantigen to the single, upper strand of NRE1 was shown to be mediated by both Ku subunits (Torrance et al, 1998). The binding of Ku to double-stranded NRE1, however, although it also requires the two subunits, occurs in a two-step manner.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

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

Ruiz

Matheos

Price

et al. 1999

MBoC

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“…Repression was also absent from cells containing inactivating mutations in the Ku80 Ϫ/Ϫ subunit but was recovered upon replenishment of Ku80 (30). Subsequently, we have reported that Ku also binds to the upper, single strand of NRE1 (upNRE1) in the MMTV LTR (78), which suggested that the effects of Ku-DNA-PK cs at NRE1 may also involve the Ku-mediated induction of a structural transition in the DNA.…”

mentioning

confidence: 91%

“…Gel slices including the Ku-DNA complexes were then excised from the gel, washed for 10 min three times in SDS sample buffer, heated to 95°C for 5 min, inserted into the wells of SDS-15% polyacrylamide gels, and electrophoresed to separate the cross-linked products. Both protocols are modifications of procedures reported previously (31,78). In the protease experiments, the control undigested samples were also cross-linked in the gel.…”

Section: Methodsmentioning

confidence: 99%

“…KMnO 4 treatment was performed as described below for covalently closed circular plasmid DNAs. Following modification, the DNA was restricted, cleaved with piperidine, and resolved on DNA sequencing gels against a KMnO 4 sequencing track of the lower, polypyrimidine-rich NRE1 strand prepared by treatment of the single strand of the parent fragment with 1 mM KMnO 4 , as we have previously described (78).…”

Section: Methodsmentioning

confidence: 99%

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Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

Giffin¹,

Gong²,

Schild-Poulter³

et al. 1999

Molecular and Cellular Biology

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Mouse mammary tumor virus (MMTV) transcription is repressed by DNA-dependent protein kinase (DNA-PK) through a DNA sequence element, NRE1, in the viral long terminal repeat that is a sequence-specific DNA binding site for the Ku antigen subunit of the kinase. While Ku is an essential component of the active kinase, how the catalytic subunit of DNA-PK (DNA-PK cs ) is regulated through its association with Ku is only beginning to be understood. We report that activation of DNA-PK cs and the repression of MMTV transcription from NRE1 are dependent upon Ku conformation, the manipulation of DNA structure by Ku, and the contact of Ku80 with DNA. Truncation of one copy of the overlapping direct repeat that comprises NRE1 abrogated the repression of MMTV transcription by Ku-DNA-PK cs . Remarkably, the truncated element was recognized by Ku-DNA-PK cs with affinity similar to that of the full-length element but was unable to promote the activation of DNA-PK cs . Analysis of Ku-DNA-PK cs interactions with DNA ends, double-and single-stranded forms of NRE1, and the truncated NRE1 element revealed striking differences in Ku conformation that differentially affected the recruitment of DNA-PK cs and the activation of kinase activity.The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) includes a complex transcriptional regulatory region that is strongly responsive to steroid hormones and prolactin (5, 6, 13-15, 18, 37, 39, 67, 73, 83). Tumorigenesis is mediated through the insertional activation of cellular protooncogenes (12,42,84). In addition to the promoter-proximal hormone response element and distal prolactin-responsive region, MMTV also contains several DNA sequence elements in the central portion of the LTR that repress or limit virally induced transcription and the response of the virus to steroid hormones (3,51,55,56,76,77,90). At least some of these elements appear to function to restrict virally induced gene expression and tumorigenesis to the lactating mammary gland, where the effects of prolactin and steroid converge to overcome the negative regulatory elements to promote a strong induction of virally induced transcription. Deletion of portions of the viral LTR that include one or more of the negative regulatory sequences deregulates MMTV-induced transcription and leads to virally induced tumors at sites not normally observed with wild-type virus, most notably T-cell lymphoma (40,77,90,91).Studies by several groups have shown that one of the negative regulatory sequences in the MMTV LTR that acts to repress viral transcription occurs in a region between 350 and 400 bp upstream of the viral transcriptional initiation site (21,40,77,85,90). We previously delimited the repressor element within this region, NRE1, to 23 bp of DNA centered over a sequence containing an overlapping direct repeat of the sequence GAGAAAGA (31). NRE1 was shown to inhibit transcription from the MMTV promoter-proximal regulatory region in T cells and in transformed mammary fibroblasts derived from an MMTV-induced tumor (31). De...

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“…In addition to DNA ends, Ku is capable of binding artificial DNA structures containing single-to double-strand transitions (15,18). There are also certain sequences that appear to function as entry sites for Ku in double-stranded DNA (19).…”

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confidence: 99%

Photocross-linking of an Oriented DNA Repair Complex

Yoo

Kimzey²,

Dynan

1999

Journal of Biological Chemistry

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Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively.

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Sequence-specific Binding of Ku Autoantigen to Single-stranded DNA

Cited by 22 publications

References 88 publications

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

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

Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

Photocross-linking of an Oriented DNA Repair Complex

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