1. Helicases as components of macromolecular machines 3

2. Helicases in replication 7

2.1 The loading of replicative helicases 7

2.1.1 Loading Rep helicase at the replication origin of bacteriophage ϕX174 7

2.1.2 How is a ssDNA strand passed through (and bound in?) the central channel of the hexameric replicative helicases? 8

2.1.3 Loading of E. coli DnaB helicase in the absence of an auxiliary protein-loading factor 8

2.1.4 The T7 gp4 primase-helicase is loaded by means of a facilitated ring-opening mechanism 10

2.1.5 Bacteriophage T4 gp61 primase can be viewed as a loading factor for the homologous gp41 helicase 11

2.1.6 DnaC serves as the loading factor for E. coli DnaB helicase 11

2.1.7 The role of bacteriophage T4 gp59 in loading the T4 gp41 helicase 12

2.1.8 Loading of helicases onto ssDNA covered by ssDNA-binding proteins (SSBPs) 15

2.2 DNA polymerase and ssDNA-binding proteins can serve as reporters for replicative helicases in their elongation mode 17

2.2.1 The DNA polymerase, the sliding clamp, and the clamp loader 17

2.2.2 The role of ssDNA-binding protein 18

2.2.3 Coupling is achieved by the DNA polymerase and the ssDNA-binding protein 18

2.3 Arrest of replicative helicases 18

2.3.1 The Ter sites and termination proteins 19

2.3.2 Models for orientation-specific fork arrest 20

3. Helicases in transcription 20

3.1 Assisted loading of E. coli RNAP by the sigma70 initiation factor 21

3.1.1 RNAP holoenzyme formation 23

3.1.2 Formation of closed promoter complexes RPc and RPi 24

3.1.3 Strand separation and the formation of the open complex 24

3.1.4 Promoter clearance 24

3.1.5 Conclusions 25

3.2 Transcript formation serves as a monitor (reporter) of RNAP helicase activity in the elongation phase of transcription 25

3.2.1 Structural aspects of transcription complex translocation 26

3.2.2 Transcript elongation is an approximately isoenergetic process 26

3.3 Termination of transcription 27

3.3.1 Intrinsic termination 27

3.3.2 Termination by transcription-termination helicase Rho 28

3.3.3 Conclusions 29

3.4 Loading of the Rho transcription-termination helicase 29

4. Helicases in nucleotide excision repair (NER) 30

4.1 The limited strand-separating activity of the UvrAB complex 31

4.2 UvrB is a DNA helicase adapted for NER 33

4.2.1 The ATP-binding site of UvrB is similar to that of other helicases 33

4.2.2 The putative DNA-binding site 33

4.3 UvrA as a UvrB loader 34

4.4 Assisted targeting of UvrAB to the transcribed strand of DNA sequences undergoing active transcription 34

4.4.1 Targeting of UvrAB to damaged DNA sites in the vicinity of promoters is assisted by RNAP 34

4.4.2 TRCF participates in the assisted targeting of UvrAB to a transcribing RNAP stalled by a DNA lesion 35

4.4.3 Conclusions 36

4.5 UvrC endonuclease is the reporter of UvrAB helicase activity in incision 36

4.6 Post-incision events 36

4.7 Mechanistic details of the helicase activity of UvrD 37

4.7.1 Structural organization and conformational changes 37

4.7.2 Translocation and unwinding activities 38

4.7.3 Step size of DNA unwinding 38

4.7.4 Oligomeric state 39

5. Helicases in recombination 39

5.1 Role of RecBCD and RecQ in the initiation of recombination 40

5.1.1 The RecBCD enzyme 40

5.1.1.1 Loading of RecBCD onto its DNA substrate does not require a separate loading protein 40

5.1.1.2 The endonuclease activity of RecD, and the binding of SSB protein, serve as reporters of RecBCD helicase activity 40

5.1.1.3 RecA can also serve as a reporter of RecBCD helicase activity 41

5.1.1.4 RecBCD step size and unwinding mechanism 41

5.1.1.5 RecBCD unwinding efficiency 42

5.1.2 The RecQ protein 43

5.2 Strand-exchange reaction catalyzed by RecA 43

5.2.1 The nucleoprotein filament 44

5.2.2 The strand-exchange reaction 46

5.2.2.1 A ‘minor-groove’ to ‘major-groove’ triple-helix transition 46

5.2.2.2 Role of the secondary DNA-binding site of RecA 46

5.2.2.3 SSB protein stimulates the strand-exchange reaction 46

5.2.2.4 Cost of the strand-exchange reaction 47

5.2.3 Conclusion: RecA is a ‘scaffolding’ protein that prepares DNA for a coupled unpairing–reannealing reaction 48

5.3 Role of the RuvAB helicase in processing recombination intermediates by a branch migration mechanism 48

5.3.1 A brief description of the RuvA and RuvB proteins 49

5.3.2 Crystal structures of RuvA and the RuvA–Holliday junction complex 50

5.3.3 RuvA as a scaffolding protein that prepares the homoduplex for strand separation 51

5.3.4 Branch migration mechanism 51

6. RNA unwindases in the spliceosome 52

6.1 RNA structural rearrangements within the spliceosome: an overview 52

6.2 The spliceosome consumes chemical free energy 54

6.3 RNA structural alterations require the concerted (or coupled) action of unwinding and reannealing proteins 54

6.4 The reannealing proteins of the spliceosome: contribution of the RNA recognition motifs (RRMs) 55

6.5 The RNA unwindases of the spliceosome 55

6.6 RNA targets of the RNA unwindases 56

7. Conclusions and overview 57

8. Acknowledgments 58

9. References 59

In Part I of this review [Delagoutte & von Hippel, Quarterly Reviews of Biophysics (2002) 35, 431–478] we summarized what is known about the properties, mechanisms, and structures of the various helicases that catalyze the unwinding of double-stranded nucleic acids. Here, in Part II, we consider these helicases as tightly integrated (or coupled) components of the various macromolecular machines within which they operate. The biological processes that are considered explicitly include DNA replication, recombination, and nucleotide excision repair, as well as RNA transcription and splicing. We discuss the activities of the constituent helicases (and their protein partners) in the assembly (or loading) of the relevant complex onto (and into) the specific nucleic acid sites at which the actions of the helicase-containing complexes are to be initiated, the mechanisms by which the helicases (and the complexes) translocate along the nucleic acids in discharging their functions, and the reactions that are used to terminate the translocation of the helicase-containing complexes at specific sites within the nucleic acid ‘substrate’. We emerge with several specific descriptions of how helicases function within the above processes of genetic expression which, we hope, can serve as paradigms for considering how helicases may also be coupled and function within other macromolecular machines.

1. Introduction 71

2. Electron transfer in PS II 72

3. (Mn)4cluster and mechanism of water oxidation 73

4. Organization and structure of the protein subunits 75

5. Organization of chlorophylls and redox active cofactors 81

6. Implications arising from the structural models 82

7. Perspectives 84

8. Acknowledgements 86

9. Addendum 86

10. References 87

Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3·7/3·8 Å, have allowed assignments to be made to the redox active cofactors involved in the light-driven water–plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the D1 protein and approximately 7 Å from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.

1. Introduction 92

2. Methods and models 93

2.1 Density Functional Theory 93

2.2 Chemical models 98

3. Examples of mechanisms studied 104

3.1 Photosystem II 105

3.2 Cytochrome c oxidase 108

3.3 Manganese catalase 112

3.4 Ribonucleotide reductase 114

3.5 Methane mono-oxygenase 119

3.6 Methyl coenzyme M reductase 122

3.7 Intra- and extradiol dioxygenases 124

3.8 Tyrosinase and catechol oxidase 126

3.9 Amino-acid hydroxylases 130

3.10 Isopenicillin N synthase 132

3.11 Cytochrome c peroxidase 134

3.12 Copper-dependent amine oxidase 136

3.13 Galactose oxidase 138

4. Summary and conclusions 138

5. Acknowledgements 140

6. References 140

The study of metalloenzymes using quantum chemical methods of high accuracy is a relatively new field. During the past five years a quite good understanding has been reached concerning the methods and models to be used for these systems. For systems containing transition metals hybrid density functional methods have proven both accurate and computationally efficient. A background on these methods and the accuracy achieved in benchmark tests are given first in this review. The rest of the review describes examples of studies on different metalloenzymes. Most of these examples describe mechanisms where dioxygen is either formed, as in photosystem II, or cleaved as in many other enzymes life cytochrome c oxidase, ribonucleotide reductase, methane mono-oxygenase and tyrosinase. In the descriptions below high emphasis is put on the actual determination of the transition states, which are the key points determining the mechanisms.