Heat-shock proteins associated with base excision repair enzymes in HeLa cells.
Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.
Mendez
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F
Departments of Radiation Oncology and Radiology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210th Street, Bronx, New York 10467, USA.
Sandigursky
M
M
Franklin
W A
WA
Kenny
M K
MK
Kureekattil
R
R
Bases
R
R
D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D019951:DNA Polymerase beta / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D009363:Neoplasm Proteins; D009994:Osmolar Concentration; D011233:Precipitin Tests; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D055551:HSP27 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011960:Receptors, Estrogen; D004247:DNA; D019951:DNA Polymerase beta; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase