Helicases as Molecular Motors BIOC/MMG 352 Scott Morrical Dept. of Biochemistry <smorrica@uvm>

1. Helicases as Molecular Motors BIOC/MMG 352 Scott Morrical Dept. of Biochemistry <smorrica@uvm.edu>

2. References Levin MK and Patel, SS (2003) “Helicases as Molecular Motors”, Chapter 7 in Molecular Motors (M. Schliwa, ed.), WILEY-VCH Verlag GmbH & Co, KgaA, Wenheim, pp. 179-203. Eoff RL and Raney KD (2005) “Helicase-catalysed translocation and strand separation”, Biochemical Society Transactions 33, 1474-1478. Korolev S, Yao N, Lohman TM, Weber PC, Waksman G (1998) “Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases. Protein Sci. 7, 605-610. Lohman TM, Thorn K, and Vale RD (1998) “Staying on track: common features of DNA helicases and microtubule motors”, Cell 93, 9-12.

3. Helicases are… …linear motor proteins that couple energy from NTP hydrolysis to translocate along a polynucleotide lattice. DNA helicases RNA helicases RNA/DNA helicases “Screwdrivers of the cell”

4. DNA Helicases Translocate on DNA lattices. Unwind duplex DNA to form ssDNA intermediates required for DNA replication, recombination, repair. Process, translocate branched DNA structures-- Holliday junctions, D-loops, etc. DNA Translocases: Translocate w/o unwinding.“DNA pumps”-- conjugation, viral packaging. Turnover of protein-DNA complexes.

5. RNA Helicases Translocate on RNA lattices. Destabilize RNA secondary structure; promote ribosome assembly, translation, RNA splicing, editing, transport, & degradation. RNA/DNA Helicases Unwind RNA/DNA hybrids; transcription termination, regulate DNA replication initiation, etc.

6. Helicases & Human Disease

7. Also… Many viruses encode their own specific helicases.

8. DNA Replication Fork DNA Helicases in the Replisome DNA Flow in the E. coli Replisome

9. Replisome in Motion (zoom out)

10. Replisome in Motion (zoom in)

11. equals DNA Helicases in Recombination Stalled DNA replication forks Holliday junction RuvAB* *E. coli RuvAB Holliday junction translocase

12. Translocation of Holliday Junctions by RuvAB Helicase in Homologous Recombination & DNA Repair RuvB hexamer RuvB hexamer RuvA tetramer

13. Basic Properties of Helicases Nucleic acid-stimulated NTPase (Mg++ dependent) Unidirectional translocation and unwinding: 5’ --> 3’ or 3’ --> 5’ Oligomeric structure (or lack thereof): some are monomeric molecular motors dimers, hexamers common other uncommon forms observed

14. NTP NDP Helicase Polarity Most helicases require a stretch of ssNA of specific polarity adjacent to the duplex region to initiate strand separation. 5’ --> 3’ helicase: 5’ 5’

15. Rarely helicases can unwind from a blunt end: 5’ NTP NDP NTP NDP RecBCD 3’ i.e. E. coli RecBCD enzyme which has 2 helicase subunits (one 3’ --> 5’ and one 5’ --> 3’) that can simultaneously engage both 5’ and 3’ ended strands from a blunt dsDNA end. Helicase Polarity (cont’d) Some helicases require both 5’ and 3’ non-complementary tails to initiate unwinding: 5’ 5’ 3’ 3’ (This is common among hexameric ring helicases found at replication forks.)

16. Helicase Superfamilies Amino acid sequence homology reveals conserved helicase motifs including Walker A and B (motifs I and II, respectively). Variations allow grouping into several related families and superfamilies: SF1 SF2-- DEAD, DEAH, DexH families named for variations in Walker B RecQ SF3 Hexameric ring-- 6 identical (most) or non-identical (MCM helicases) subunits Family members probably share a general mechanism of translocation and unwinding; however there is no correlation between sequence and polarity or substrate specificity.

17. Helicase Structure • All helicases share a RecA fold. • Each helicase molecule contains a single NTP binding site and a distinct polynucleotide binding site. • These sites are allosterically linked, since the NTPase activity modulates nucleic acid binding affinity, and vice-versa.

18. Structure of Hepatitus C Virus (HCV) Helicase (SF2) 1A 2A 3 1A 2A 3

19. Rep (SF1) vs. HCV (SF2) Helicases Conservation of structure and relationships of conserved helicase motifs despite little overall sequence homology. Rep HCV

20. Sequence Alignments of the SF1 and SF2 Superfamilies Based on Structural Super-Imposition

21. Common Features of SF1 and SF2 Helicase Structure • Sub-domains 1A and 2A contain RecA-like folds. • Residues from conserved helicase motifs line the interface of 1A and 2A and bind NTP. • ssNA binds in a groove that is formed by 1A and 2A sub-domains. • Motifs I, II, and III form NTP binding site while motifs V and VI are positioned so as to help transmit a conformational change from the NTP binding site to the NA binding site. • Domain movements between 1A and 2A as well as rigid body rotations of other sub-domains have been implicated as intermediates in helicase mechanisms.

22. Structure of Helicase Domain of Bacteriophage T7 gp4 Helicase

23. T7 gp4 Helicase Structure (cont’d) (C) Stereo view from inside the ring of T7 gp4 helicase subunits A and B complexes to ADPNP (dark blue) and Mg++ (green, space filling). The helicase conserved motifs are shown in the same color as in (B). The subunits are colored the same as in (A). Amino acid residue R522 is shown.

24. Common Features of Ring Hexamer Helicase Structures • Helicase domain fold is RecA-like. • Conserved helicase motifs and NTP binding site are at subunit interface. • A critical arginine residue (R522 in T7 gp4 helicase) from a neighboring subunit is within hydrogen-bonding distance of the gamma phosphate of NTP bound at the interface, and is implicated in transducing conformational changes between subunits of the hexamer. • The central channel of the ring is large enough to accommodate a single strand of DNA or RNA. • Rings appear to adopt multiple asymmetric conformations in response to ligand binding.

25. Mechanisms of Unidirectional Translocation & Unwinding • Unwinding is a combination of unidirectional translocation and strand separation processes. • Both processes require energy from NTP hydrolysis. • NTP binding, hydrolysis, and product release act as a switch that induces conformational changes on the helicase NA binding site. • Conformational changes force the helicase to alter its NA-binding affinity or to perform a power stroke. • Conformational changes drive unidirectional translocation and unwinding in a step-wise fashion.

26. Mechanisms of Unidirectional Translocation & Unwinding: Stepping Models Stepping requires at least 2 NA binding sites that independently bind and release NA and change the distance between each other. • Monomeric helicases: 2 hands represent 2 parts of NA binding site that move relative to each other. Both sites are controlled by a single NTPase site. • Oligomeric helicases: hands represent NA binding sites on 2 different subunits. Coordinated NTPase activity at multiple sites leads to coordinated binding/release of NA.

27. Mechanisms of Unidirectional Translocation & Unwinding: Stepping Models (cont’d) • Helicase undergoes a round of conformational changes (i to vi) after which it appears in the same conformational state (i’) one step away from its original position. • Cycle starts with 1st NA binding site tightly bound to NA (closed hand, i) and 2nd site weakly bound to NA (open hand). • In a power stroke motion, the 2nd site moves away from the 1st (ii), followed by tight binding of the 2nd site (closing, iii), opening of the 1st site (iv), a power stroke bringing sites back together (v), closing of the 1st site (vi), and finally opening of the 2nd site (i’).

28. A Stepping Model Proposed for Dimeric Rep Helicase Note that Rep actively engages duplex to promote unwinding.

29. Mechanisms of Unidirectional Translocation & Unwinding: Brownian Motor Use thermal fluctuations from surroundings to achieve unidirectional movement, biased by NTP hydrolysis. • Helicase has a periodic depen-dence of binding energy along the length of ssNA (saw-tooth) • If saw-tooth is asymmetric as shown, then most helicase-ssNA binding events (i) will cause left-to-right movement, bringing helicase to its local minimum (ii) where it is trapped.

30. Mechanisms of Unidirectional Translocation & Unwinding: Brownian Motor (cont’d) Use thermal fluctuations from surroundings to achieve unidirectional movement, biased by NTP hydrolysis. • Now suppose that NTP binds and lowers helicase-ssNA binding energy and makes it uniform along the length of the ssNA (dotted line). • This allows the helicase to slide along ssNA randomly in either direction because of Brownian motion (iii & iv). • After NTP hydrolysis and product release, the asymmetric saw-tooth profile returns and helicase binds to ssNA tightly. If Brownian motion moved the helicase backward (iii) it will end up in the same position it started in and there is no net movement (ii). • However if Brownian motion moves the helicase forward (iv) it will end up one step forward after NTP hydrolysis and product release (ii’).

31. Factors Influencing Brownian Motor Efficiency Efficiency = fraction of productive steps / NTP hydrolyzed • The greater the assymetry of the sawtooth, the greater the efficiency, since it will increase the fraction of helicase molecules that reach poisiton iv and make a step forward. • Efficiency depends strongly on rate of ATP hydrolysis, which determines the lifetime of the weakly bound state (iii & iv). • A helicase can move forward even against a moderate external force as long as a significant fraction of the helicase can diffuse forward to position iv.

32. Stepping vs. Brownian Motors • Stepping requires at least 2 independent NA binding sites that move relative to each other. • A Brownian motor with only 1 NA binding site can still move unidirectionally. • Stepping-- force is generated at the junction of the NA binding sites. This force moves the two binding sites relative to each other, generating unidirectional movement. • Brownian-- 2 kinds of forces: One is generated at the NA binding site during tight binding of helicase to NA. The other is Brownian motion that can move the helicase in any direction along the NA while the helicase binding is in a flat energy profile. • Brownian model predicts limited processivity for monomeric helicases. • Dimeric or hexameric helicases with 2 or 6 ssNA binding sites can implement a Brownian mechanism more efficiently, especially if NTPase cycles are coordinated between subunits.

33. Mechanisms of Unidirectional Translocation & Unwinding Estimating Step Size and Processivity • The step size of a helicase can be estimated from single turnover kinetics of unwinding reactions and fitting to a stepping equation. • The lower limit step size can be determined by measuring the coupling ratio (number of bases traveled per NTP hydrolyzed). • Processivity (P) = probability of making a step forward on a NA lattice divided by the probability of dissociation from that position on the lattice. • Monomeric helicases: UvrD, PcrA P ≈ 0.9 (10 bp) • Hexameric helicases: T7 gp4 P ≈ 0.99996 (33 kbp)

34. Mechanisms of Unidirectional Translocation & Unwinding Strand Separation Basepair hydrogen bond free energies: DG ≈ 10 kJ mol-1 base-1 at 15ºC (unwinding thermodynamically unfavorable) Spontaneous opening rates: 1000 s-1 near ends 30 s-1 in middle

35. Mechanisms of Unidirectional Translocation & Unwinding Strand Separation via “Active” vs. “Passive” Mechanisms • To make unwinding thermodynamically favorable, helicases must stabilize open bps by binding to ssNA one step at a time. • Active mechanisms: helicase increases the rate of bp opening in addition to stabilizing the ssNA product. • Rate can be increased by lowering transition state energy. • Specific binding to unwinding intermediate, i.e. distorted duplex region. • Passive mechanisms: by definition can’t change rate of bp opening. May be useful for helicases with large step size.

36. Mechanisms of Unidirectional Translocation & Unwinding Another View: Helicase as Translocating Wedge • A helicase could in principle unwind dsNA by translocating between 2 strands of NA while pushing them apart without any specific interactions. • Any protein capable of translocating along one strand of dsNA while excluding the other can unwind dsNA by a wedge mechanism (see RuvAB).

37. T7 gp4 helicase shows little interaction with duplex, unlike Rep or PcrA. A classic wedge? Note small step size (est 2 bp).

38. Replisome in Motion (zoom in)