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A structural feature of Dda helicase which enhances displacement of streptavidin and trp repressor from DNA.

ABSTRACT: Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nearly wild type activity for unwinding double-stranded DNA but exhibits greatly reduced streptavidin displacement activity. Interestingly, this domain has little effect on displacement of either gp32 or BamHI bound to DNA but does affect displacement of trp repressor from DNA. With this variant, we have identified residues which enhance displacement of some proteins from DNA.

SUBMITTER: Byrd AK

PROVIDER: S-EPMC8819844 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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A structural feature of Dda helicase which enhances displacement of streptavidin and trp repressor from DNA.

Byrd Alicia K AK Malone Emory G EG Hazeslip Lindsey L Zafar Maroof Khan MK Harrison David K DK Thompson Matthew D MD Gao Jun J Perumal Senthil K SK Marecki John C JC Raney Kevin D KD

Protein science : a publication of the Protein Society 20211122 2

Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nea ...[more]

PMID: 34761452

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Project description:Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription, and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called nonstructural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains, respectively. The helicase domain of NS3 is termed NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3'-5' direction. The directionality of unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5' end. NS3h can bind to the ssDNA and in the presence of ATP move toward the 5' end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h and 200 nM oligonucleotide), we find that NS3h translocates on ssDNA at a rate of 46 +/- 5 nucleotides/s, and that it can move for 230 +/- 60 nucleotides before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5' end. Upon addition of streptavidin, a "protein block" was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single-cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase in its monomeric form, as has been reported. In summary, NS3h translocates on ssDNA as a monomer, but the translocase activity does not correspond to comparable DNA unwinding activity or protein displacement activity under identical conditions.

Dataset Information

A structural feature of Dda helicase which enhances displacement of streptavidin and trp repressor from DNA.

Publications

A structural feature of Dda helicase which enhances displacement of streptavidin and trp repressor from DNA.

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