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)