RecA family protein mediated homologous recombination

RecA-family proteins play vital roles in DNA repair and sexual reproduction.  By combining single molecule force experiments, MD simulations, analytical modeling, and bulk experiment the Prentiss group has greatly enhanced understanding of the structure/function relationships during homologous recombination.   In RecA mediated homologous recombination, the binding of RecA to a  single stranded DNA (ssDNA) forms an ssDNA-RecA filament that searches double stranded DNA ( dsDNA  ) for a sequence region that corresponds to the sequence of the ssDNA in the ssDNA-RecA filament.   Using single molecule force experiments, the Prentiss group showed that homology recognition can be disabled by pulling on the ends of the dsDNA. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287184/ They also did analytical modeling suggesting that the stress on dsDNA bound to RecA could limit the number of bp that could be probed during the initial homology search and could drive strand exchange forward. https://journals.aps.org/pre/abstract/10.1103/PhysRevE.87.032702

A combination of bulk experiments, molecular modeling, and analytical modeling revealed the early steps in strand exchange and showed that the initial homology test considers 8 bp and accepts one mismatch. https://pubmed.ncbi.nlm.nih.gov/26089391/ https://pubmed.ncbi.nlm.nih.gov/26384422/.

A summary of the results is available at https://www.youtube.com/watch?v=uKymU6kkoRs.

                

 

Experimental work by the Greene group at Columbia, and the Haber group at Brandeis demonstrated that homology recognition in yeast also begins with an 8 bp test that accepts one mismatch.

Originally, people believed that since RecA is an ATPase, ATP hydrolysis must be required for homology recognition; however, pioneering work showed that homology recognition can occur without ATP hydrolysis.  The Prentiss group showed that though homology recognition of 20 bp sequences can proceed without ATP hydrolysis,  ATP hydrolysis is required to provide homology recognition for sequences longer than ~30 bp. https://pubmed.ncbi.nlm.nih.gov/28854739/  Furthermore, collaboration with the Prevost group provided structural insight into the ATP hydrolysis induced instability that provides homology recognition for sequences longer than 30 bp.

https://academic.oup.com/nar/article/47/15/7798/5542884?login=true

Surface view of two turns of RecA filaments
Surface view of two turns of RecA filaments built on a DNA single-strand with (A) a regular ATP-binding geometry and (B) a distorted geometry obtained when replacing the central interface by an ADP-binding geometry, with a second (blue) and third (cyan) strand of DNA added.

 

To complete dsDNA repair, if the RecBCD pathway is followed RecA-mediated homologous recombination must be followed by DNA synthesis by a DNA polymerase.