Calf thymus DNA helicase F, a replication protein A copurifying enzyme.
A DNA helicase from calf thymus, called DNA helicase F, copurified with replication protein A through several steps of purification including DEAE-Sephacel, hydroxyapatite and single stranded DNA cellulose. It is finally separated from replication protein A on FPLC Mono Q where the DNA helicase elutes after replication protein A. Characterization of the DNA helicase F by affinity labeling with [alpha 32P]ATP indicated that the enzyme has a catalytic subunit of 72 kDa. Gel filtration experiments suggested that DNA helicase F can exist both in a monomeric and an oligomeric form. The enzyme unwinds DNA in the 5'-->3' direction in relation to the strand it binds. All eight deoxyribonucleoside- and ribonucleosidetriphosphates could serve as an energy source. Testing a variety of DNA/DNA substrates demonstrated that the DNA helicase F preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail. However, replication protein A allowed the DNA helicase to unwind much longer DNA substrates of up to 400 bases, indicating that the copurification of replication protein A with the DNA helicase F might be of functional relevance.
Georgaki
A
A
Department of Veterinary Biochemistry, University of Zürich-Irchel, Switzerland.
Tuteja
N
N
Sturzenegger
B
B
Hübscher
U
U
D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins; D011506:Proteins; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases