Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro.
Human single-stranded DNA-binding protein (HSSB, also called RPA), is a heterotrimeric complex that consists of three subunits, p70, p34, and p11. HSSB is essential for the in vitro replication of SV40 DNA and nucleotide excision repair. It also has important functions in other DNA transactions, including DNA recombination, transcription, and double-stranded DNA break repair. The p34 subunit of HSSB is phosphorylated in a cell cycle-dependent manner. Both Cdc2 kinase and the DNA-dependent protein kinase (DNA-PK) phosphorylate HSSB-p34 in vitro. In this study, we show that efficient phosphorylation of HSSB-p34 by DNA-PK requires Ku as well as DNA. The DNA-PK phosphorylation sites in HSSB-p34 have been mapped at Thr-21 and Ser-33. Kinetic studies demonstrated that a phosphate residue is first incorporated at Thr-21 followed by the incorporation of a second phosphate residue at Ser-33. We also identified Ser-29 as the major Cdc2 kinase phosphorylation site in the p34 subunit.
Niu
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Graduate Program in Molecular Biology, Memorial Sloan Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
Erdjument-Bromage
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H
Pan
Z Q
ZQ
Lee
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SH
Tempst
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Hurwitz
J
J
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen