A DNA helicase from Xenopus laevis ovaries

Abstract

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.

Xenopus laevis Ovarian DNA Helicase I: A 3' to 5' Helicase That Unwinds Short Duplexes

“…As shown in Figure 4, comparable amounts of MS2 RNA did not compete with the DNA helicase substrate. (Poll & Benbow, 1988 (Poll & Benbow, 1988 Activity then decreased at higher salt concentrations in parallel with DNA unwinding activity ( Figure 5B). …”

“…This possibility was further investigated by measuring thermal inactivation of the activities ( Figure 5C). (Poll & Benbow, 1988). The unwinding of longer duplex regions was investigated to determine the maximal length of fragment that was displaced.…”

“…Human DNA helicases II and IV require a single-stranded region of over 84 bases adjacent to duplex DNA to support unwinding (Tuteja et al, 1993). DNA unwinding by X laevis DNA helicase I is supported by hydrolysis of ATP or dATP, and is not supported by ATP analogs or other nucleoside triphosphates (Poll & Benbow, 1988). Addition of ATP analogs or nucleoside triphosphates to the DNA unwinding and DNA-dependent ATPase assays gives rise to complex effects (Table 1).…”

“…X. laevis ovarian DNA helicase I was purified as described by Poll and Benbow (1988). The experiments 1 Abbreviations: ssDNA, single-stranded DNA; dsDNA, doublestranded DNA; ATP7S, adenosine 5'-0-(3-thiotriphosphate); AMPPNP, adenylyl 5'-|8,7-imidodiphosphate); AMPPCP, adenylyl 5'-(/S,7-methylenediphosphonate); E. coli ssb, Escherichia coli single-strand DNA binding protein; HSSB, human single-strand binding protein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.…”

“…Thirteen picomoles of the 12-mer 5'-TCAC-GACGTTGT was hybridized to the indicated oligonucleotide (see Figure 3) in 10 jtL of 10 mM Tris-HCl (pH 8.0), 1 mM MgCl2, and 200 mM NaCl, as described above (molar ratio 1:2.5). The 12-mer primer was elongated in the presence of [a-32P] dATP and 5 units of Klenow fragment of E. coli DNA polymerase I as described (Stahl et al, 1986) (Poll & Benbow, 1988 …”