Dataset Information

Regulation of translocation polarity by helicase domain 1 in SF2B helicases.

ABSTRACT: Structurally similar superfamily I (SF1) and II (SF2) helicases translocate on single-stranded DNA (ssDNA) with defined polarity either in the 5'-3' or in the 3'-5' direction. Both 5'-3' and 3'-5' translocating helicases contain the same motor core comprising two RecA-like folds. SF1 helicases of opposite polarity bind ssDNA with the same orientation, and translocate in opposite directions by employing a reverse sequence of the conformational changes within the motor domains. Here, using proteolytic DNA and mutational analysis, we have determined that SF2B helicases bind ssDNA with the same orientation as their 3'-5' counterparts. Further, 5'-3' translocation polarity requires conserved residues in HD1 and the FeS cluster containing domain. Finally, we propose the FeS cluster-containing domain also provides a wedge-like feature that is the point of duplex separation during unwinding.

SUBMITTER: Pugh RA

PROVIDER: S-EPMC3261565 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Publications

Regulation of translocation polarity by helicase domain 1 in SF2B helicases.

Pugh Robert A RA Wu Colin G CG Spies Maria M

The EMBO journal 20111111 2

Structurally similar superfamily I (SF1) and II (SF2) helicases translocate on single-stranded DNA (ssDNA) with defined polarity either in the 5'-3' or in the 3'-5' direction. Both 5'-3' and 3'-5' translocating helicases contain the same motor core comprising two RecA-like folds. SF1 helicases of opposite polarity bind ssDNA with the same orientation, and translocate in opposite directions by employing a reverse sequence of the conformational changes within the motor domains. Here, using proteol ...[more]

PMID: 22081110

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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

Regulation of translocation polarity by helicase domain 1 in SF2B helicases.

Publications

Regulation of translocation polarity by helicase domain 1 in SF2B helicases.

Similar Datasets

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