Person:

Feinstein, Efraim

A RecA–single-stranded DNA (RecA–ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA–ssDNA filaments, whereas pulling on the two 3′, the two 5′, or all four termini does not. We propose that the ‘outgoing’ strand in the dsDNA is extended by strong DNA–protein contacts, whereas the ‘complementary’ strand is extended by the tension on the base pairs that connect the ‘complementary’ strand to the ‘outgoing’ strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present.

This paper describes an empirical model of polymer dynamics, based on the agitation of millimeter-sized polymeric beads. Although the interactions between the particles in the macroscopic model, and those between the monomers of molecular-scale polymers, are fundamentally different, both systems follow the Worm-Like Chain theory.

RecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA–ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.