Testing ancient RNA–protein interactions

The past decade in molecular biology has seen remarkable advances in the study of the origin and early evolution of life. The mathematical tools for analyzing DNA and protein sequences, coupled with the availability of complete microbial genome sequences, provide insight almost as far back as the age of the nucleic acids themselves. Experimental evolution in the laboratory and especially in vitro evolution of RNA provide insight into a hypothetical world where RNA, or a close relative, may have debuted as a primary functional and informational molecule. The ability to isolate new functional RNAs from random sequences now ultimately makes the world of possible primitive chemical interactions accessible even when the molecules or reactions are no longer present in modern species. Thus we can at last form direct experimental tests of specific models for the origin of RNA–protein associations, such as those that influenced the genetic code. This marks a turning point for probing the origin and early history of life at the molecular level.

Topological testing of the mechanism of homology search promoted by RecA protein

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA–single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA–ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA–ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA–ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson–Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA–ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.