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Helicase mechanisms and the coupling of helicases within macromolecular machines Part I: Structures and properties of isolated helicases

Published online by Cambridge University Press:  17 February 2003

Emmanuelle Delagoutte
Affiliation:
Institute of Molecular Biology and Department of Chemistry, University of Oregon Eugene, OR 97403, USA
Peter H. von Hippel
Affiliation:
Institute of Molecular Biology and Department of Chemistry, University of Oregon Eugene, OR 97403, USA

Abstract

1. Mechanisms of nucleic acid (NA) unwinding by helicases 432

2. Helicases may take advantage of ‘breathing’ fluctuations in dsNAs 434

2.1 Stability and dynamics of dsNAs 434

2.2 dsNAs ‘breathe’ in isolation 435

2.3 Thermodynamics of terminal base pairs of dsNA 438

2.4 Thermal fluctuations may be responsible for sequential base-pair opening at replication forks 439

2.5 Helicases may capture single base-pair opening events sequentially 440

3. Biochemical properties of helicases 443

3.1 Binding of NAs 443

3.2 Binding and hydrolysis of NTP 445

3.3 Coordination between NA binding and NTP binding and hydrolysis activities 446

4. Helicase structures and mechanistic consequences 447

4.1 Amino-acid sequence analysis reveals conserved motifs that constitute the NTP-binding pocket and a portion of the NA-binding site 447

4.2 Organization of hepatitis virus C NS3 RNA helicase 449

4.2.1 Biochemical properties of HCV NS3 449

4.2.2 Crystal structures of HCV NS3 helicase 450

4.2.2.1 The apoprotein 450

4.2.2.2 The protein–dU8 complex 450

4.2.3 A possible unwinding mechanism 452

4.2.4 What is the functional oligomeric state of HCV NS3? 452

4.3 Organization of the PcrA helicase 453

4.3.1 The apoenzyme and ADP–PcrA complex 454

4.3.2 The protein–DNA–sulfate complex 456

4.3.3 The PcrA–DNA–ADPNP complex 456

4.3.4 A closer look at the NTP-binding site in the crystal structure of PcrA–ADPNP–DNA 457

4.3.5 Communication between domains A and B 457

4.3.6 How might ssDNA stimulate the ATPase activity of PcrA? 457

4.3.7 A possible helicase translocation mechanism 458

4.3.8 A possible unwinding mechanism 458

4.4 Organization of the Rep helicase 459

4.4.1 Biochemical properties 459

4.4.2 Crystal structure of Rep bound to ssDNA 462

4.5 Organization of the RecG helicase 462

4.6 Hexameric helicases 466

4.6.1 Insights from crystal structures of hexameric helicases 467

4.6.2 Possible translocation and unwinding mechanisms 468

5. Conclusions 469

6. Acknowledgments 472

7. References 472

Helicases are proteins that harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of double-stranded nucleic acids. These enzymes have been much studied in isolation, and here we review what is known about the mechanisms of the unwinding process. We begin by considering the thermally driven ‘breathing’ of double-stranded nucleic acids by themselves, in order to ask whether helicases might take advantage of some of these breathing modes. We next provide a brief summary of helicase mechanisms that have been elucidated by biochemical, thermodynamic, and kinetic studies, and then review in detail recent structural studies of helicases in isolation, in order to correlate structural findings with biophysical and biochemical results. We conclude that there are certainly common mechanistic themes for helicase function, but that different helicases have devised solutions to the nucleic acid unwinding problem that differ in structural detail. In Part II of this review (to be published in the next issue of this journal) we consider how these mechanisms are further modified to reflect the functional coupling of these proteins into macromolecular machines, and discuss the role of helicases in several central biological processes to illustrate how this coupling actually works in the various processes of gene expression.

Type
Research Article
Copyright
© 2002 Cambridge University Press

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