Lysogeny maintenance: a matter of looping Laura Finzi

1. Lysogeny maintenance: a matter of looping Laura Finzi

2. Two possible modes LYSOGENIC MODE (passive replication) LYTIC MODE (active replication) Lytic mode Lysogenic mode replication l is a temperate phage EFFICIENT REGULATION OF GENIC EXPRESSION

3. 0 minutes 30 minutes 45 minutes Lytic cycle

4. l repressor (CI) is responsible for maintenance of lysogeny λ CI protein acts both as a transcriptional activator and as a repressor in the maintenance of the lysogenic cycle Ptashne M., 1992, “A genetic switch”, Cambridge, MA: Blackwell Scientific Pubblications and Cell Press

5. Loop-based model of the l repressor auto-regulation (or how to maintain the perfect concentration) The occupancy of OR3 by CI and, consequently, the mechanism of negative autoregulation, depend on the interaction among CI molecules bound to the OL and OR regions, about 2.4 kbp apart Revet B. et al. Current Biology 9:151-154, 1999. / Dodd I.B. et al. Genes and development 15:3013-3022, 2001. / Dodd I.B. et al. Genes and development 18:344-354, 2004.

6.  Motion amplitude, (nm)   Time (s) TPM-Protein-induced dynamic DNA looping produces a telegraphic-like signal  D. Schafer et al. Nature 352:444, 1991 / L. Finzi & J. Gelles Science 267:378, 1995 P. Nelson, et al., “Tethered Particle Motion as a Diagnostic of DNA Tether Length”, The Journal of Physical Chemistry B, 110, 17260, 2006

7. Data aquisition and analysis (1) Labview routine Drift subtraction DIC image x and y coordinates x’ and y’ coordinates of the anchor point ρ┴(t) = [(x(t)-x’)2+(y(t)-y’)2]1/2 Plot of ρ┴ over time P. Nelson, et al., “Tethered Particle Motion as a Diagnostic of DNA Tether Length”, The Journal of Physical Chemistry B, 110, 17260, 2006

8. Dig Bio OL1 OL2 OL3 OR3 OR2 OR1 302 – 306 – 1051- 2317 bp DelOL1 DelOL1,2 DelOL1-3 Bio Dig OL2 OL3 OR3 OR2 OR1 Bio Dig OL3 OR3 OR2 OR1 Bio Dig OR3 OR2 OR1 DNA fragments used in the TPM measurements • -Verified loop formation in the DNA mediated by CI bound to the OL and OR regions • Determined the relative importance of the three OL operators in loop formation • Determined the effect of the distance between the OL and OR regions C. Zurla, et al., “Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda” Journal of Physics: Condensed Matter, 18, S225-S234, 2006.

9. Wt loop formation and breakdown

10. Dig Bio OL1 OL2 OL3 OR3 OR2 OR1 302 – 306 – 1051- 2317 bp DelOL1 DelOL1,2 DelOL1-3 Bio Dig OL2 OL3 OR3 OR2 OR1 Bio Dig OL3 OR3 OR2 OR1 Bio DNA fragments used in the TPM measurements Dig OR3 OR2 OR1 • -Verified loop formation in the DNA mediated by CI bound to the OL and OR regions • Determined the relative importance of the three OL operators in loop formation • Determined the effect of the distance between the OL and OR regions C. Zurla, et al., “Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda” Journal of Physics: Condensed Matter, 18, S225-S234, 2006.

11. Effect of progressive deletions of the OL operators Del OL1 Del OL1,2 Del OL1-3 control 20 nM 100 nM control 20 nM 100 nM control 20 nM 40 nM 100 nM C. Zurla, et al.,J.P.C.M.18, S225-S234, 2006.

12. Dig Bio OL1 OL2 OL3 OR3 OR2 OR1 302 – 306 – 1051- 2317 bp DelOL1 DelOL1,2 DelOL1-3 Bio Dig OL2 OL3 OR3 OR2 OR1 Bio Dig OL3 OR3 OR2 OR1 Bio Dig OR3 OR2 OR1 DNA fragments used in the TPM measurements • -Verified loop formation in the DNA mediated by CI bound to the OL and OR regions • Determined the relative importance of the three OL operators in loop formation • Determined the effect of the distance between the OL and OR regions C. Zurla, et al., “Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda” Journal of Physics: Condensed Matter, 18, S225-S234, 2006.

13. Loop probability analysis: loop size effect ; 302 bp 1051 bp U C U U L L 2317 bp L 100 nM control 20 nM control 100 nM control 100 nM C. Zurla, et al.,J.P.C.M.18, S225-S234, 2006. Dt

14. PRM expression titration

15. Looping Titration (CI nM) 0 100 200 20 400 50

16. Dwell times distributions for unlooped and looped states at [CI] = 50 and 200 nM

17. Summary of wild type lifetimes

18. Summary • Loop formation probability increases with CI protein concentration. • There are multiple unlooped and looped species. Hypothesis: 1) Closure of the loop can be mediated by DNA/CI complexes containing different numbers of CI dimers (different occupation/loading levels) each with different stability.

19. Possible configurations 1 9 6 18 9 6 6 18 2 9 9 6 6 6 1 1 49 64

20. OL3 OL2 OL1 OL3 OL2 OL1 PL PL PR PR PRM OR3 OR2 OR1 PRM OR3 OR2 OR1 Summary • Loop formation probability increases with CI protein concentration. • There are multiple unlooped and looped species. Hypothesis: 1) Closure of the loop can be mediated by DNA/CI complexes containing different numbers of CI dimers (different occupation/loading levels) each with different stability. 8mer – pRM is transcribed 12mer – all the promoters are repressed • Competition experiments, • Measurements with point-mutated operators. • DHMM analysis (J.F. Beausang et al., BJ-BLetters, DNA looping kinetics analyzed using DHMM, 2007) may help characterize hidden intermediates.

21. Effect of [CI] on the equilibrium constant of the looping reaction(Kloop) Kloop = Dloop/Dunloop = total time spent in the looped config./total time spent in the unlooped configuration

22. Dwell times distributions for unlooped and looped states in the presence of 200 nM CI and 10,000X competitor DNA Single exponential

23. Point-mutated operators 1 2 3 3 2 1 x O1- x x x O2- x O3- x LR

24. Probability distribution of <r>in mutated DNA fragments mutated DNA: 4 operators available wt-DNA: 6 operators

25. Lifetimes: oL1-oR1-

26. Lifetimes: oL2-oR2-

27. Lifetimes: oL3-oR3-

28. Summary of mutant lifetimes

29. x x O3- x x x x x x x O1- x x x O2- x x x x Interpretation for O3- single exponential L 1 2 3 R 3 2 1 R L

30. Estimation of ∆Gloop For each measured looping equilibrium an expression can be developed in terms of [CI] and free energy, leaving looping free energies as fit parameters.

31. Population probability and estimation of ∆Gloop GL1 = GL1, binding GL1,2 = GL1, binding + GL2,binding + GL1,2,coop GLoop1,2 = GL1, binding + GL2,binding + GL1,2,coop + GR1, binding + GR2,binding + GR1,2,coop + Gloopoctamer

32. ∆Gloop pi, loop / pi, unlooped Black: wt DNA, Blue: O1-, Red: O2- Green: O3- ∆GLOOP tetramer: 1 kcal/mol ∆GLOOP octamer: 0.8 kcal/mol ∆GLOOP dodecamer: -0.5 kcal/mol

33. Dodecamer precursor L R 1 2 3 3 2 1

34. Conclusions • CI mediates a loop between the L and R region of  DNA • OL1 and OL2 are critical for loop formation • Wild type loop is quite dynamic • Probability of loop formation increases with CI concentration • There are multiple possible looped species • Competition and point mutated operators (O3-) allow detection of octamer-mediated loop • Population probability analysis indicates that octamer-mediated loop is quite unstable and suggests a decamer might be the precursor

35. What about DNA supercoiling?

36. Gal repressor requires supercoiling  + 0.03 0 - 0.015 250nm - 0.03 - 0.06 50s 100s 0s Time Lia et al, PNAS 2003

37. Hat curve for wt  DNA fragment Fragment length: ~ 11,000 bp

38. 0.64 microns Force-jump experiments yield loop lifetime

39. Shorter loop shows transitions Fragment length: ~ 4900 bp Loop size: 393 bp

40. Force and supercoiling oppose looping Force Negative supercoiling

41. Conclusions • We observed CI-mediated wild type loop formation and measured loop • We can measure the effect of tension and supercoiling on a short CI-mediated loop • Level of supercoiling is critical for the stability of the loop • More experiments are needed!!!!

42. Contributors • Emory University • Chiara Zurla • Carlo Manzo • Laura Finzi • David Dunlap • NCI, NIH • Dale Lewis • Sankar Adhya • University of Pennsylvanya • John Beausang • Phil Nelson Support: HFSP, Emory University Research Council