Purification and characterization of delta helicase from fetal calf thymus.
A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.
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Department of Medicine, University of Miami School of Medicine, Florida 33101.
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D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010777:Photochemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013950:Thymus Gland / Q000196:embryology / Q000201:enzymology*
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases