Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication.
Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum ... binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.
Mesh Terms:
Animals, Antibodies, Antigens, Nuclear, Cricetinae, Cricetulus, DNA Helicases, DNA Replication, DNA-Binding Proteins, DNA-Directed DNA Polymerase, Deoxyribonucleases, Type II Site-Specific, Haplorhini, Hela Cells, Host Cell Factor C1, Humans, Nuclear Proteins, Octamer Transcription Factor-1, Oligonucleotides, Replication Origin, Replication Protein A, Transcription Factors
Animals, Antibodies, Antigens, Nuclear, Cricetinae, Cricetulus, DNA Helicases, DNA Replication, DNA-Binding Proteins, DNA-Directed DNA Polymerase, Deoxyribonucleases, Type II Site-Specific, Haplorhini, Hela Cells, Host Cell Factor C1, Humans, Nuclear Proteins, Octamer Transcription Factor-1, Oligonucleotides, Replication Origin, Replication Protein A, Transcription Factors
Biochim. Biophys. Acta
Date: Oct. 11, 2002
PubMed ID: 12393188
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