Monomer Structure
is one of the many proteins that is involved in recombination cross-over events and during recombination repair in response to single strand DNA breaks. RecA is a rather small monomer protein that can multiplex with itself up to thousands of RecA proteins to associate with ssDNA. The structure of RecA was determined through x-ray crystallography and each monomer contains very distinct structural components. These are a largely helical 30-residue N-terminal region, a 240-residue α/ß ATPase core, and a 64-residue C-terminal
globular domain.
The process of recruiting new RecA monomers is carried out through an ATP-dependent process. This occurs through the binding of ATP to two adjacent on subsequent RecA monomers. To properly grow the crystal that was used to determine structure through x-ray crystallography, a non-hydrolyzable analog of ATP is used. This analog has the shorthand formula of ADP-AlF4-Mg. Specifically, the aluminum tetrafluoride is bound to the adenine diphosphate in the ɣ position. Several residues are involved in the hydrolysis of ATP to coordinate strand exchange after binding to ssDNA has occurred. On one of the RecA monomers, two lysine residues, Lys 248 and Lys 250, are responsible for coordinating with the ɣ phosphate and stabilizing it. Lys 250 has also been implicated to have an additional function: to coordinate a glutamic acid, Glu 96, on the adjacent RecA monomer. This coordination with Glu 96 is achieved through hydrogen bonding and is believed to be critical for the catalytic mechanism. Specifically, there is a very complex network of hydrogen bonding that is occurring between several other residues to rotate Glu 96 to a more favorable conformation enabling Glu 96 to act as a nucleophile. (ask how much detail about this is necessary. Could potentially be another scene)
Once several RecA monomers have coordinated with one another, they coordinate with ssDNA to form a repeating structure that contains exactly three nucleotides for every RecA monomer. However, this does not mean that each nucleotide triplet only interacts with a single RecA monomer. In reality, each RecA monomer spans three nucleotides, but the nucleotide triplet interacts with the other two RecA surrounding it in both the 5' and 3' direction. Essentially, each nucleotide triplet is interacting with three different RecA monomers named RecA5', RecA0, and RecA3' based on their relative location to the nucleotide triplet. The first nucleotide of the triplet is bound by both RecA5' and RecA0, the second is bound only by Rec0 and the third is bound by both Rec0 and Rec3'. is responsible for stabilizing ssDNA within this conformation. Specifically, the phosphate backbone of the nucleotide triplet is what interacts with the RecA monomer residues through hydrogen bonding (dashed lines). Interestingly, the hydrogen bonding interactions that are occurring do not always use the side chains, but often will interact with the amide groups on amino acid backbones. For example, the first phosphate group within a nucleotide triplet will interact with the backbone amide of Met 197 from RecA5' and the amide backbone of Asn 123 from RecA0. The second phosphate of the triplet interacts with Gly 211 and Gly 212 on RecA0. The third phosphate of the triplet is unique as it interacts with the side chains of Ser 172 and Arg 176.
Once a RecA filament has properly formed and coordinated with ssDNA, a complementary DNA strand must be located. Once a complementary strand is located, the donor dsDNA is wound into the filament complex where the ssDNA and dsDNA form a temperate three-stranded DNA intermediate. Another protein complex, RecBCD, not modeled here, helps resolve the strand exchange process via the formation of a holiday junction. The process of strand exchange is heavily mediated through traditional Watson-Crick base pairing rules, but also by a few residues located within the RecA filament complex. Specifically, Ser 162 on each RecA monomer contacts the phosphate groups near the nucleotide triplet. Additionally, Met 164 increases the spacing between nucleotide triplets by inserting itself into the gap between them. This insertion allows for more strict base pairing stabilization. The final residue implicated in strand exchange appears to have a proofreading mechanism similar to that of DNA polymerases. This residue is Arg 169 and has been implicated in having base-pairing proofreading abilities by hydrogen bonding with O2 groups in thymidine bases. This interaction is able to check for proper Watson-Crick base pairing because the bond lengths associated with proper base pairing will allow for proper interactions between Arg 169 and thymidine. Incorrect base pairing will cause thymidine residues to shift position preventing necessary Arg 169 interactions. This functionality has been shown through the mutation of Arg 169 to Histidine resulting in ultraviolet sensitivity and increased mismatched base pairing.
