Dissecting Tn5 transposition using HIV-1 integrase diketoacid inhibitors

Area of Science:

• Molecular Biology
• Enzymology
• Biochemistry

Background:

• Diketoacid (DKA) compounds inhibit HIV-1 integrase by sequestering active site metals.
• HIV-1 integrase and Tn5 transposase share similar active site architectures and catalytic mechanisms.

Purpose of the Study:

• Investigate DKA analogues' inhibition of Tn5 transposase activity.
• Utilize Tn5 transposase as a model system to explore DKA inhibitor mechanisms.

Main Methods:

• Screened several hundred DKA analogues for activity against Tn5 transposase.
• Assessed DKA compound effects on various transposition steps, including paired end complex (PEC) formation, DNA cleavage, and strand transfer.
• Utilized active site mutations and donor DNA to probe compound binding sites.

Main Results:

• Identified several DKA analogues with activity against Tn5 transposase.
• All tested DKA compounds inhibited PEC formation; some decreased PEC stability.
• Four DKA compounds inhibited DNA cleavage, with one preferentially inhibiting the second cleavage step.
• All six compounds inhibited strand transfer, with two showing no effect on DNA cleavage.
• Inhibition of strand transfer was sensitive to the 5'-non-transferred DNA strand structure.

Conclusions:

• DKA inhibitors exhibit varied effects on Tn5 transposition, suggesting distinct binding interactions.
• The differential inhibition of cleavage steps indicates a sequential process requiring conformational changes.
• DKA compounds provide valuable tools for mechanistic studies of transposases and integrases.