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

Poll, E. H. A.; Harrison, Jeff; Umthun, Angela R.; Dobbs, Drena; Benbow, Robert M.

doi:10.1021/bi00179a007

Cited by 4 publications

(3 citation statements)

References 35 publications

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“…A possible candidate for the unwinding of the hairpins with 3 0 -tails in the Xenopus egg extracts would be Xenopus ovarian helicase I (Poll & Benbow, 1988). This 3 0 -5 0 helicase requires a single-stranded 3 0 tail of at least 4 to 9 nt adjacent to the duplex to be unwound in order to enter its substrate and it unwinds only short duplexes of about 16 bp (Poll et al 1994). The low efficiency of the unwinding of duplexes longer than 16 bp could account for the observed incomplete cleavage of hairpins with 3 0 -tails (duplex stem length of 37 bp) by the denuclease: inefficient unwinding of these substrates would render only part of the 5 0 recessed ends accessible to cleavage by the decanuclease.…”

Section: Discussionmentioning

confidence: 99%

“…The products of the first cleavage step again would require unwinding so that the newly created cleavable fraction could fall below the detection limit. The abovementioned features of the Xenopus ovarian 3 0 -5 0 helicase I to unwind only short duplexes with high efficiency (Poll et al 1994) could account for this phenomenon.…”

Section: Discussionmentioning

confidence: 99%

“…This 3 0 -5 0 helicase requires a single-stranded 3 0 -tail of at least 4 to 9 nt adjacent to the duplex to be unwound in order to enter its substrate and it unwinds only short duplexes of about 16 bp (Poll et al 1994). The products of the first cleavage step again would require unwinding so that the newly created cleavable fraction could fall below the detection limit.…”

Section: Discussionmentioning

confidence: 99%

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**A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA**

et al. 1996

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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**A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA**

et al. 1996

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Organization, Replication, Transposition, and Repair of DNA

Metzler¹,

Metzler²,

Sauke³

2001

Biochemistry

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DNA Helicase Activity of the Hepatitis C Virus Nonstructural Protein 3

Gwack

Kim

Han³

et al. 1997

European Journal of Biochemistry

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Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA . DNA and RNA . RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA-stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3' to 5'. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall order of inhibitory capacity was : adenosine 5'-0-(3-thiotriphosphate) > adenylyl-imidodiphosphate and adenylyl-(b,y-methy1ene)-diphosphate > AMP. DNA helicase activity was inhibited strongly by ssDNA and ssRNA, but was little affected by dsDNA. The ATPase activity was stimulated greatly by ssDNA and ssRNA, but not by dsDNA. The NS3 protein could unwind up to 500 base pairs of duplex DNA.The possible multifunctional nature of the NS3 protein is discussed and compared with that of Simian virus 40 large T antigen.Keywords: non-structural protein 3 ; hepatitis C virus ; DNA helicase; RNA helicase ; processivity.Hepatitis C virus (HCV) was first isolated in 1989 and is the major etiologic agent of transfusion non-A, non-B hepatitis [ 11. HCV is an enveloped virus that contains a positive-strand RNA genome of about 9400 nucleotides. The RNA genome of HCV contains a single open reading frame that encodes a viral polyprotein of about 3010 amino acids. This polyprotein is processed by viral and host proteases into the C, El, E2, NS2, NS3, NS4A, NS4B, NSSA, and NSSB proteins [2-51. The amino-terminal domain of the NS3 protein contains a trypsin-like serine proteinase activity, which cleaves the NS3-NS4A, NS4A-NS4B, NS4B-NSSA, and NSSA-NSSB protein junctions of the HCV polyprotein [6].HCV belongs to the positive-strand RNA virus family Flaviviridae. The other two members of this family are flavivirus and pestivirus 171. Comparative sequence analyses of these three viruses reveal distinct similarities [8]. In particular, the HCV NS3 protein and its homologs from the other Flaviviridae family members contain the conserved sequence motifs of RNA helicases and NTPases [9-111. The NTPase activities of NS3 and its homologous proteins display several similar characteristics, for example, optimal pH, optimal magnesium concentration, sensitivity to monovalent cations, and relaxed substrate specificity (they can utilize both NTPs and dNTPs) [12, 131.The carboxyl-terminal domain of the HCV NS3 protein shares sequence similarity with the DEAD box family of RNA Kan...

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

Cited by 4 publications

References 35 publications

**A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA**

**A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA**

Organization, Replication, Transposition, and Repair of DNA

DNA Helicase Activity of the Hepatitis C Virus Nonstructural Protein 3

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