DNA Strand Exchange

Barcode-based self-assembly

For the purpose of analyzing the homology search and recognition process in DNA, the structure of dsDNA can be modeled as a complex structure of self-assembling building blocks.  Prentiss Research Group analyzes various modes of self-assembly which are reflective of the biological homolog pairing process, and which might be used to aid synthetic self-assembly of barcode-tagged parts.

One possible strategy for self-assembling a complex structure utilizing building blocks is to try many structures and rapidly disassemble those that are erroneous (non-homologous) until the correct structure begins to form, and then converge rapidly to the complete correct structure.  We focus our numerical studies on linear structures that self-assemble due to an attractive potential resulting from individual elements, or bits, that form a bar code that identifies the different structures. With many possible interactions, many energy minima exist in the energy landscape of homology levels, producing incorrectly bound conformations.  We find that long, rigid structures easily become trapped in false energy minima.  In contrast, if the structure is composed of small rigid sections separated by flexible neutral regions, as in the ssDNA-RecA-dsDNA triplex interaction, the likelihood of successful self-assembly is vastly increased.  Biology may take advantage of this strategy in homolog pairing.

 


 

Homology search and sequence matching in dsDNA

Homology_Search

Features of Homology Recognition and Strand Exchange

  1. Testing is an iterative process that progresses through contiguous basepairs
    1. Avoids kinetic trapping in short regions of accidental homology
  2. Testing is done on small groups (~3) where the entire group tests together and assembles or disassembles together
    1. Increases sequence stringency by amplifying the free energy cost of a mismatch
  3. Testing begins with a minimal length of ~ 9 basepairs
    1. Avoids kinetic trapping in short regions of accidental homology
  4. Testing includes at least 2 states, one of which is not the same as the assembled state
    1. Allows the initial  testing state to be free energetically unfavorable, which drives rapid unbinding of mismatched sequences
    2. Allows the initial testing state to be a non-linear function of the number of basepairs in the testing state, which limits the maximum sequence length that can test without doing strand exchange, which reduces kinetic trapping
  5. The decrease in free energy due to correct matching is a non-linear function of the number of contiguous assembled bases
    1. Allows sequences with lengths<N to be unstable, while sequences with lengths >N are very stable, as required for accurate homology recognition of sequences with length N

 

Structure of strand-exchange protein

The processes of sexual reproduction and DNA repair both require DNA strand exchange, wherein a single-stranded DNA (ssDNA) is able to locate and pair with a sequence-matched double-stranded DNA molecule (dsDNA).  These strand exchange processes rely on proteins which act as universal mediators for recombination.  Though some features of these homologous recombination and damage-induced repair processes are well known, many of the details that underlie rapid and accurate sequence recognition are unclear.

Strand_Exchange

 

To probe the functionality of strand exchange proteins, we focus on exploration of RecA, a strand exchange protein found in E. coli bacteria.  In strand exchange, RecA proteins first bind along ssDNA via the protein’s primary site.  The resulting filament binds with the outgoing strand and its complementary strand of via the protein’s secondary site to form a ssDNA-RecA-dsDNA filament (A).  Base pairs from the complementary strand of the dsDNA base-flip from the outgoing strand in the secondary site toward the incoming ssDNA strand, forming a double-strand in the protein’s primary site (B).  If the base-pairs are non-homologous, the unfavorable pairing drives the complementary strand to return to the protein’s secondary site, reforming the initial double-strand and allowing the protein to unbind.  If homology is encountered, strand exchange may proceed further along the dsDNA.  If an incorrect pairing is encountered, the incoming single strand and the RecA proteins are released.  This process facilitates the repair of non-complementary regions in dsDNA which is otherwise largely homologous.