DOC PREVIEW
Berkeley MCELLBI 110 - Crystal Structures of Complexes of PcrA DNA Helicase

This preview shows page 1-2-3 out of 10 pages.

Save
View full document
View full document
Premium Document
Do you want full access? Go Premium and unlock all 10 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 10 pages.
Access to all documents
Download any document
Ad free experience
View full document
Premium Document
Do you want full access? Go Premium and unlock all 10 pages.
Access to all documents
Download any document
Ad free experience
Premium Document
Do you want full access? Go Premium and unlock all 10 pages.
Access to all documents
Download any document
Ad free experience

Unformatted text preview:

Cell, Vol. 97, 75–84, April 2, 1999, Copyright 1999 by Cell PressCrystal Structures of Complexes of PcrA DNAHelicase with a DNA Substrate Indicatean Inchworm Mechanismbetween domains 1A and 2A that is lined with a numberof conserved sequence motifs that are characteristicof helicases (Gorbalenya and Koonin, 1993). PcrA is amember of a large family of helicases that have a 39–59directionality (Bird et al., 1998a) and share a number ofSameer S. Velankar,†Panos Soultanas,†Mark S. Dillingham,†Hosahalli S. Subramanya,and Dale B. Wigley*Sir William Dunn School of PathologyUniversity of Oxfordstructural features (Bird et al., 1998b). Other membersSouth Parks Roadof this family include the Rep and UvrD helicases, whichOxford OX1 3REhave been the subject of a great deal of study in recentUnited Kingdomyears (reviewed in Lohman and Bjornson, 1996), and theNS3 RNA helicase from hepatitis C virus (Yao et al.,1997; Cho et al., 1998; Kim et al., 1998; Porter et al.,Summary1998).The crystal structure of Rep helicase complexed withWe have determined two different structures of PcrAsingle-stranded DNA (Korolev et al., 1997) provided theDNA helicase complexed with the same single strandfirst insights into the interaction of the protein with DNA.tailed DNA duplex, providing snapshots of differentIntriguingly,theenzymecrystallized intwo differentcon-steps on the catalytic pathway. One of the structuresformations (termed “open” and “closed”), with the twois of a complex with a nonhydrolyzable analog of ATPmolecules sitting adjacent to each other on the single-and is thus a “substrate” complex. The other structurestranded dT(pT)15oligonucleotide. The conformationalcontains a bound sulphate ion that sits in a positiondifference between the molecules comprised a largeequivalent to that occupied by the phosphate ion pro-rigid body rotation of the 2B domain by approximatelyduced after ATP hydrolysis, thereby mimicking a “prod-1308. This “domain swiveling” was proposed to be anuct” complex. In both complexes, the protein is mono-important aspect of the mechanism of the enzyme. Sur-meric. Large and distinct conformational changes occurprisingly, although Rep has been reported to be a dimeron binding DNA and the nucleotide cofactor. Takenin the presence of single-stranded DNA (Chao and Loh-together, these structures provide evidence againstman, 1991), the protein proved to be monomeric in thean “active rolling” model for helicase action but arecrystal structure. The only other helicase for which thereinstead consistent with an “inchworm” mechanism.are structural data is the NS3 RNA helicase (Yao et al.,1997; Cho et al., 1998; Kim et al., 1998). The structureshows a tandem repeat of domains, each with foldsIntroductionsimilar to domains 1A and 2A but with a slightly differentconnectivity (Bird et al., 1998b). The third domain of theHelicases are found in all living organisms and partici-protein has no structural homology with Rep or PcrApate in almost every process that involves nucleic acidsbut sits in a position roughly equivalent to that occupied(Lohman and Bjornson, 1996). Sequence analysis ofby domains 1B and 2B in the closed conformation ofbacterial genomes has revealed that even in these or-Rep. The structure of a complex of the protein with aganismsthereis arequirement foraroundadozendiffer-(dU)8oligonucleotide (Kim et al., 1998) showed theent helicases. The biochemical activity of the enzymesssRNA-bindingsiteto belocated inapositionequivalentis to couple the free energy of hydrolysis of ATP to theto the binding site for ssDNA in the Rep helicase. Threeseparation of a DNA (or RNA) duplex into its componentdifferent models for the mechanism of the enzyme havestrands. Although the physiological role of some heli-been proposed, one to accompany each of the threecases has been determined, the functions of many oth-structures. Biochemical and structural data show theers remain unclear. One such example is PcrA helicaseenzyme to be monomeric under a range of differentthat, although shown to be an essential enzyme in Bacil-conditions (Yao et al., 1997; Cho et al., 1998; Kim et al.,lussubtilis (Petitetal., 1998)and Staphylococcus aureus1998; Porter et al., 1998).(Iordanescu, 1993) involved in repair and rolling circleThere are two popular models for a general mecha-replication (Petit et al., 1998; Soultanas et al., 1999),nism for helicases (reviewed in Bird et al., 1998b) (Figurehas an imprecisely defined physiological role in cells. In1), termed the “inchworm” (Yarranton and Gefter, 1979)order to learn more about this enzyme, we have initiatedor “active rolling” models (Wong and Lohman, 1992),a study of PcrA helicase from the moderate thermophilerespectively. Experiments to distinguish conclusivelyBacillus stearothermophilus and have reported the pre-between these models have been difficult to design,liminary characterization of the enzyme (Bird et al.,largely because the two models actually share a number1998a) and its crystal structure (Subramanya et al.,of similarities in terms of the biochemical events taking1996). The enzyme comprises four domains termed 1A,place, and most experimental observations are in fact1B, 2A, and 2B, with domains 1A and 2A having veryconsistent with either mechanism. There are, however,similar folds. The ATP-binding site is situated in a clefta few features distinct to each mechanism. One of themost important of these is the absolute requirementof the rolling model for (at least) a dimeric protein. By*To whom correspondence should be addressed (e-mail: wigley@contrast, the inchworm model is consistent with anyeric.path.ox.ac.uk).†These authors contributed equally to this work.oligomeric state for the protein, including monomeric.Cell76Results and DiscussionStructure of a Complex with DNA and a SulphateIon—A Product ComplexInitial attempts to solve the structure of the PcrA/DNA/sulphate complex by molecular replacement using theapo enzyme structure as a starting model proved to beunsuccessful, the first indication that a conformationalchange hadtaken place. The best startingmodelprovedto be one that combined just domains 1A and 2A—domains 1B and 2B had to be located separately. Theoverall fold of the protein is the same as that describedpreviously for the apo protein (Subramanya et al., 1996),but the domain orientations are very different (Figure 2).Although domains 1A and 2A are in similar orientationsFigure 1. Active


View Full Document

Berkeley MCELLBI 110 - Crystal Structures of Complexes of PcrA DNA Helicase

Documents in this Course
Midterm

Midterm

7 pages

Midterm

Midterm

5 pages

Exam

Exam

15 pages

Load more
Download Crystal Structures of Complexes of PcrA DNA Helicase
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Crystal Structures of Complexes of PcrA DNA Helicase and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Crystal Structures of Complexes of PcrA DNA Helicase 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?