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.