Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.

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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Mammalian polynucleotide kinases (PNKs) carry out 5'-phosphorylation of nucleic acids. Although the cellular function(s) of these enzymes remain to be delineated, important suggestions have included a role in DNA repair and, more recently, in DNA replication. Like T4 PNK, some preparations of mammalian PNKs have been reported to have an associated 3'-phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3'-phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI-PNK, with a slightly acidic pH optimum that copurifies with a 3'-phosphatase activity. The enzyme appears to be a monomer of 60 kDa. Mammalian DNA replication reactions were supplemented with T4 PNK or SNQI-PNK, and no significant effect on DNA replication in vitro was observed. Database searches support the earlier mapping of the 3'-phosphatase activity of T4 PNK to the C-terminus and suggest that the 3'-phosphatase domain of T4 PNK is related to the protein superfamily of L-2-haloacid dehalogenases. Exopeptidase digestion experiments were carried out to compare the SNQI-PNK enzyme with T4 PNK and led to the inference that the domain organization of the bovine polypeptide may differ from that of the T4 enzyme.