RecBCD - Department of Molecular & Cell...
Transcript of RecBCD - Department of Molecular & Cell...
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RecBCD
Joshua AdelmanMCB 290
Walking Helicases II
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Outline• Brief overview of helicase families
• RecBCD
• Structure
• Observing single molecule translocation
• Stopped flow probe of RecBCD mutants
• Chi as a molecular throttle
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Overview of Helicases/Translocases/Remodelases
Borrowed without permission from J. Berger’s MCB 200 Lecture
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SF1/SF2 Catalytic Site Organization
PcrA
Walker A/ P-Loop
Walker B
Arg-Finger
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Variation in walking motif structures
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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)
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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)
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RecBEndonuclease + 3’ to 5’ Helicase
2B
1B
‘Arm’
2A
1A
Singleton, et al Nature 432, 187-193 (11 November 2004)
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RecB5’ to 3’ Helicase
‘2A’ ‘1A’
Contains disordered domain that corresponds to 2B
Singleton, et al Nature 432, 187-193 (11 November 2004)
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RecDnonfunctional SF1 homologue
Processivity Factor?Singleton, et al Nature 432, 187-193 (11 November 2004)
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Chi Site Activation
Singleton, et al Nature 432, 187-193 (11 November 2004)
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Observing Single RecBCD Activity
Bianco, et al. Nature 409, 374-378 (18 January 2001)
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Observing Single RecBCD Activity
Bianco, et al. Nature 409, 374-378 (18 January 2001)
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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
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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
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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.
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Chi sequence as a molecular throttle
Spies, et al (2003) Cell 115(5)647-654
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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.
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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
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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
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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)