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TRANSCRIPT
RecBCD
Joshua AdelmanMCB 290
Walking Helicases II
Outline• Brief overview of helicase families
• RecBCD
• Structure
• Observing single molecule translocation
• Stopped flow probe of RecBCD mutants
• Chi as a molecular throttle
Overview of Helicases/Translocases/Remodelases
Borrowed without permission from J. Berger’s MCB 200 Lecture
SF1/SF2 Catalytic Site Organization
PcrA
Walker A/ P-Loop
Walker B
Arg-Finger
Variation in walking motif structures
Double Strand Break RepairRecBCD loads onto a blunt double strand break
ATP dependent translocation coupled to nuclease activity
Chi-site recognition changes activity and mediates RecA loading
Singleton, et al Nature 432, 187-193 (11 November 2004)
Structure of RecBCD
Two SF1 domains pull DNA through two channels and break it on a molecular pin.
Singleton, et al Nature 432, 187-193 (11 November 2004)
RecBEndonuclease + 3’ to 5’ Helicase
2B
1B
‘Arm’
2A
1A
Singleton, et al Nature 432, 187-193 (11 November 2004)
RecB5’ to 3’ Helicase
‘2A’ ‘1A’
Contains disordered domain that corresponds to 2B
Singleton, et al Nature 432, 187-193 (11 November 2004)
RecDnonfunctional SF1 homologue
Processivity Factor?Singleton, et al Nature 432, 187-193 (11 November 2004)
Chi Site Activation
Singleton, et al Nature 432, 187-193 (11 November 2004)
Observing Single RecBCD Activity
Bianco, et al. Nature 409, 374-378 (18 January 2001)
Observing Single RecBCD Activity
Bianco, et al. Nature 409, 374-378 (18 January 2001)
What are the roles of RecB and RecD?
- Tracking unwinding using stopped-flow dye displacement- Use dyes that have large fluorescence increase when bound to dsDNA
Dillingham, et al. J. Biol. Chem., Vol. 280, Issue 44, 37069-37077, November 4, 2005
WT RecBCDno chi-site
What are the roles of RecB and RecD?
Dillingham, et al. J. Biol. Chem., Vol. 280, Issue 44, 37069-37077, November 4, 2005
Mutate out ATPase activity of individual subunit, but retain ability to bind DNA
RecBCD mutants are effective helicases
Dillingham, et al. J. Biol. Chem., Vol. 280, Issue 44, 37069-37077, November 4, 2005
RecB and RecD also work as monomers, but are much slower and less processive.
Chi sequence as a molecular throttle
Spies, et al (2003) Cell 115(5)647-654
Chi sequence as a molecular throttle
Spies, et al (2003) Cell 115(5)647-654
- If RecBCD misses the first chi site, it can recognize the second.- RecBCD does not change behavior if it has already engaged a chi site.
Chi sequence as a molecular throttle
Pause time proportional to distance from free end to chi site
Spies, et al (2003) Cell 115(5)647-654
Chi Regulated Translocation
RecD faster than RecB(a previous study had suggested RecD was faster in disagreement with stopped-flow measurements
Translocation at the same
velocity
Spies, et al (2003) Cell 115(5)647-654
Unanswered Questions- Step size (suggested to be 1bp/2ATP or 1bp/1ATP in single subunit ATPase mutants).- Coordination of subunits: highly variable translocation rates in and between studies makes data hard to compare.- Effects of load on the motor (8 pN causes backsliding)