The DNA-rearranging IS621 recombinase inserts segments 2 000 times more often than it excises them

The DNA-rearranging IS621 recombinase inserts segments 2 000 times more often than it excises them

On August 12, Nature published a paper explaining why the IS621 recombinase inserts DNA segments more readily than it excises them. The authors traced this imbalance to the shape of the protein, RNA, and DNA complex. They then increased the frequency of excision in E. coli plasmids by modifying the guide RNA.

IS621 is a mobile DNA segment in the IS110 family. Its encoded recombinase first excises the segment from DNA to form a circular double-stranded molecule, then inserts that molecule at a new site. A specialized guide RNA called bridge RNA provides the protein with both addresses: its two loops bind the segment being transferred and the target site. A 2024 study showed that these addresses can be changed. The new paper explains why the reverse reaction is much less efficient.

In an experiment using purified protein, defined DNA segments, and an engineered bridge RNA, excision produced 0,03% product, whereas insertion produced 63%. DNA labels showed where the loss of efficiency occurred. Intermediate molecules accumulated after the first cut in both reactions, but the next step, in which the cleaved DNA strands exchange positions, was much less efficient during excision.

Cryo-electron microscopy, which reveals the structure of the protein, RNA, and DNA complex, showed why strand exchange proceeds differently in the two reactions. During insertion, the two DNA segments bend into a U shape. During excision, they form a straight cross. The authors propose that excision is less efficient because this cross-shaped arrangement makes it harder for the cleaved strands to exchange positions.

The same step is also influenced by two short nucleotide pairs, which are pairs of the chemical letters that make up RNA and DNA. During insertion, strand exchange increases the number of these pairs. Excision works better when their distribution is reversed. In E. coli plasmids, an engineered bridge RNA with altered nucleotide pairs and guide regions increased the frequency of excision by approximately 13 000 times. Increasing production of the original RNA produced roughly a hundredfold increase, whereas increasing production of the engineered RNA had almost no additional effect.

In this plasmid system, the bridge RNA sequence was the main determinant of excision frequency. The structures identified which RNA changes help the reaction complete strand exchange.