UvrD helicase activation by MutL involves rotation of its 2B subdomain.

Area of Science:

• Molecular biology
• Biochemistry
• DNA repair mechanisms

Background:

• Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in DNA repair, replication, and recombination.
• UvrD requires self-assembly or accessory proteins for in vitro helicase activity, with E. coli MutL previously shown to activate monomeric UvrD.

Purpose of the Study:

• To elucidate the mechanism by which E. coli MutL activates the UvrD helicase.
• To investigate the role of the UvrD 2B subdomain in MutL-mediated activation.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) to monitor conformational changes in the UvrD 2B subdomain.
• Kinetic studies to correlate MutL-induced conformational changes with helicase activity.
• Utilizing chimeric UvrD constructs to assess subdomain specificity.

Main Results:

• The UvrD 2B subdomain exists in an equilibrium between open and closed states when bound to DNA, favoring the open conformation.
• MutL binding shifts the 2B subdomain to an intermediate conformational state, crucial for helicase activation.
• MutL failed to activate a chimeric UvrD with a Rep helicase 2B subdomain, highlighting specificity.

Conclusions:

• MutL activation of monomeric UvrD helicase activity is specifically regulated by the UvrD 2B subdomain.
• The conformational transition of the 2B subdomain to an intermediate state is a key step in the MutL activation pathway.