Homologous recombination (HR) contributes to the maintenance of genome integrity among all kingdoms of life and serves as a driving force in evolution. During HR, a single-stranded DNA (ssDNA) is paired with the complementary strand of a homologous dsDNA, resulting in displacement of the non-complementary strand. HR plays essential roles in double-strand DNA break (DSB) repair, the rescue of stalled or collapsed replication forks, chromosomal rearrangements, horizontal gene transfer, and meiosis. 

The Rad51/RecA family of recombinases are highly conserved ATP-dependent proteins that form helical filaments on DNA and promote the transactions that take place during HR. The protein participants, nucleoprotein structures, and general reactions mechanisms are broadly conserved. Interestingly, most eukaryotes have two Rad51/RecA family recombinases: Rad51, which is constitutively expressed; and Dmc1, which is only expressed during meiosis. The Rad51 and Dmc1 lineages within the Rad51/RecA family of recombinases arose early in the evolutionary history of eukaryotes, and these proteins remain closely related; for instance,S. cerevisiae Rad51 and Dmc1 share 45% sequence identity and 56% sequence similarity and both proteins perform the same basic biochemical function, namely the pairing of homologous DNA sequences. However, it remains unclear why eukaryotes have evolved two recombinases. The primary goal of our research was to explore the biophysical and biochemical differences between Rad51 and Dmc1 in a further effort to understand why most eukaryotes utilizes two recombinases.  

Our NSF-supported research has made several key insights into the mechanistic differences between Rad51 and Dmc1, particularly with respect to their interactions with DNA substrates and protein accessory factors. For example, we have shown that Rad51 and Dmc1 respond differently to recombination intermediates bearing mismatched base pairs: Rad51 cannot stabilize mismatched base pairs, whereas Dmc1 can. This differential response may have important biological implications in that the function of Rad51 is to fix broken DNA as accurately as possible using a template DNA molecule that is identical to the broken DNA molecule. In contrast, Dmc1 has to perform recombination during meiosis between DNA molecules that may contain different parental alleles which are not exact sequence matches. Our data imply that Dmc1 is “tuned” to interact with these types of inexact DNA pairing interactions which will allow it to function correctly when promoting recombination between DNA from “mom” and “dad”. Moreover, we show that Rad51 and Dmc1 lineage–specific amino acids (i.e. amino acids that are unique to the Rad51 lineage or the Dmc1 lineage of the Rad51/RecA family of DNA recombinase) in DNA–binding loops 1 and 2 are responsible for this differential response to mismatched base pairs. These studies initially focused initially upon yeast Rad51 and yeast Dmc1, and we also extended the studies now to human Rad51 and human Dmc1, as well as C. elegans RAD-51; the significance of this last organism, is that C. elegans only has a gene for RAD-51, but our work now shows that it contains Dmc1-specific amino acids in DNA- binding loop 1. In addition to the lineage specific amino acids, we have identified in DNA–binding loops 1 and 2, we have now moved on to identify Rad51 and Dmc1 lineage–specific acids throughout the entire lengths of the two proteins (approximately 50 amino acids each in S. cerevisiae Rad51 and Dmc1) and we are working to experimentally validate these amino acids. 

We also have also that Rad51 and Dmc1 interact differently with various helicase proteins that act as potent regulators of recombination. These studies emerged from our initial observation that the S. cerevisiae Sf1 helicase Srs2 readily strips Rad51 from ssDNA at a rate of up to 50 Rad51 monomers per second, thus acting as a strong negative regulator of homologous recombination. In striking contrast, Srs2 is completely unable to remove Dmc1 from ssDNA. We have also shown that two yeast Sf2 helicases Rad54 and Rdh54 interact strongly with Rad51-ssDNA filaments and serve to inhibit the negative regulation capacity of Srs2. We believe that this observation suggests that Srs2 acts on early recombination intermediates to prevent premature recombination from taking place but is then itself downregulated upon the arrival of Rad54 and Rdh54. Thus, the regulatory activity of Srs2 with respect to Rad51 is likely limited to an early time window prior to maturation of a fully formed Rad51-ssDNA presynaptic complex. We have also extended these observations to members of the RecQ family of DNA helicases, including the yeast RecQ helicase Sgs1, and the human RecQ helicases RecQ5 and BLM. Interestingly, we find that Sgs1 and RecQ5 both readily strip Rad51 from ssDNA, and can also remove Dmc1 from ssDNA, albeit at a slower rate compared to Rad51. In striking contrast, we find that BLM helicase cannot act upon Rad51- or Dmc1-ssDNA intermediates, nor can it act upon Rad51- or Dmc1-dsDNA intermediates and instead appears to act in more specialized settings involving naked dsDNA substrates. 

Last Modified: 04/10/2024
Modified by: Eric C Greene