Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta.
Activator 1 (A1) is a multiprotein complex which is essential for proliferating cell nuclear antigen (PCNA)-dependent DNA polymerase delta (pol delta) activity and efficient in vitro DNA synthesis in the SV40 dipolymerase replication system. In this report, we describe the isolation of A1 from HeLa cytosolic extracts. A1 stimulated pol delta activity in singly primed phi X174 DNA or (dA)4500.oligo(dT)12-18 in reactions containing PCNA, single-stranded DNA binding protein (SSB), and ATP. Using this assay, A1 has been extensively purified. Purified preparations contained five discrete subunits of 145, 40, 38, 37, and 36.5 kDa. ATP hydrolysis to ADP and Pi is essential for A1-dependent pol delta activity, and we have shown that A1 contains an intrinsic ATPase which is stimulated by DNA. The DNA-dependent hydrolysis of ATP can be stimulated by PCNA and further activated by PCNA plus the human single-stranded DNA binding protein. These stimulatory effects were observed with (dA)4500.oligo(dT)12-18, but were not detected with each poly-deoxynucleotide alone. Furthermore, A1 formed a complex with (dA)4500.oligo(dT)12-18 which could be measured by nitrocellulose binding. No complex with (dA)4500 or oligo(dT)12-18 alone was detected by this procedure. Data are also presented which indicate that A1, in conjunction with PCNA, functions as a primer-recognition factor for pol delta, increasing its ability to utilize low levels of primer ends, but it does not increase the size of the DNA products. A1 also markedly reduced the amount of PCNA required for pol delta activity on a multiply primed DNA suggesting that PCNA interacts with A1 at the primer end. These multiple effects of A1 closely resemble the properties of the multisubunit protein RF-C described by Tsurimoto and Stillman (Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1023-1027).
Lee
S H
SH
Graduate Program in Molecular Biology, Sloan-Kettering Cancer Center, New York, New York 10021.
Kwong
A D
AD
Pan
Z Q
ZQ
Hurwitz
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004258:DNA Polymerase III; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun; D012265:Ribonucleotides / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D016755:Proto-Oncogene Proteins c-jun; D012265:Ribonucleotides; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases