DNA contacts stimulate catalysis by a poxvirus topoisomerase

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Eukaryotic type 1B topoisomerases manage DNA supercoiling via transient covalent enzyme-DNA intermediates.
• Poxviruses, like molluscum contagiosum, encode their own topoisomerases essential for viral replication.

Purpose of the Study:

• To investigate the DNA recognition and catalytic mechanisms of the topoisomerase from molluscum contagiosum virus.
• To identify the specific DNA sequences and protein-DNA interactions critical for enzyme activity.

Main Methods:

• DNA binding and covalent complex formation assays.
• Analysis of mutant DNA sequences and inosine-substituted DNA.
• Mapping protein-DNA contacts using chemical probes (5'-bridging phosphorothiolates).
• Kinetic studies to determine the impact of DNA sequences on catalytic steps.

Main Results:

• A larger DNA recognition site than the previously identified 5'-(T/C)CCTT-3' was observed for molluscum contagiosum virus topoisomerase.
• Distinct DNA sequences are important for initial DNA binding versus covalent complex formation.
• Flanking DNA bases influence covalent complex formation, not just initial binding.
• Specific flanking sequences accelerate catalysis by enhancing initial cleavage rather than inhibiting religation.

Conclusions:

• Unidentified DNA contacts accelerate a step between initial binding and covalent complex formation.
• These findings refine models of topoisomerase conformational changes during catalysis.
• The study provides insights into poxviral topoisomerase function and DNA-viral protein interactions.