MutL Activates UvrD by Interaction Between the MutL C-terminal Domain and the UvrD 2B Domain

• Published in: Journal of molecular biology 2024-06, Vol.436 (11), p.168589-168589, Article 168589
• Main Authors: Storozhuk, Olha, Bruekner, Susanne R., Paul, Ankon, Lebbink, Joyce H.G., Sixma, Titia K., Friedhoff, Peter
• Format: Article
• Language: English
• Subjects: cross-linking; DNA - genetics; DNA - metabolism; DNA Helicases - chemistry; DNA Helicases - genetics; DNA Helicases - metabolism; DNA mismatch repair; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; helicase; MutL Proteins - chemistry; MutL Proteins - genetics; MutL Proteins - metabolism; Protein Binding; Protein Domains; transient kinetics
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2024.168589

[Display omitted] •Combination of preparative chemical crosslinking and functional assays offers a unique strategy to determine interaction interfaces and assess functional outcomes in DNA repair processes.•Covalent attachment of MutL's CTD to UvrD 1B or 2B domain enables efficient helicase activity even on substrates with minimal 3′-tail lengths (5 nucleotides)•Persistent UvrD activity after MutL N-terminal domain removal post-crosslinking underscores the robustness of the MutLCTD-UvrD interaction.•MutL's C-terminal domain autonomously activates UvrD helicase when crosslinked to the 1B or 2B domain of UvrD. UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3′-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.