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The RecA Protein: Structure and Biological Function

Heather M. Heerssen(1), Aaron Downs(3), and David Marcey(2)

© David Marcey, 2001

I. Introduction: The Biological Function of RecA
II. Structural Overview
III. Filament Structure
IV. DNA Binding Regions of RecA

V. ATP Binding Sites

VI. Interfilament Interactions

VII. Coprotease Activity
VIII. References

Note:  This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.



I. Introduction: The Biological Function of RecA

One method by which genetic variation is generated is homologous (or general) recombination. In this process, two double stranded DNA molecules exchange segments of DNA at sites of sequence similarity through breakage and rejoining of strands, leading to recombinant chromosomes with new combinations of alleles at various genetic loci. Homologous recombination occurs in diverse organisms in different contexts, including meiotic crossing over in eukaryotes and sexduction in prokaryotes.

According to a model for homologous recombination, a nick in one of the DNA double helices (catalyzed by the RecBCD oligomeric protein in E. coli) allows a region of single stranded DNA to invade a double stranded DNA helix. The intruding strand displaces one of the helixed strands and binds to the other via Watson-Crick base pairing. The displaced strand, in turn, hybridizes with the remaining ssDNA. The regions of DNA involved in the heteroduplexes enlarges through a process called branch migration. Such "Holliday junctions" are resolved by resolvase (RuvC in E. coli) and ligase, leading to recombinant chromosomes in 50% of the resolved products.

The RecA protein is a critical enzyme in this process, as it catalyzes the pairing of ssDNA with complementary regions of dsDNA. The RecA monomers first polymerize to form a helical filament around ssDNA. During this process, RecA extends the ssDNA by 1.6 angstroms per axial base pair. Duplex DNA is then bound to the polymer. Bound dsDNA is partially unwound to facilitate base pairing between ssDNA and duplexed DNA. Once ssDNA has hybridized to a region of dsDNA, the duplexed DNA is further unwound to allow for branch migration. RecA has a binding site for ATP, the hydrolysis of which is required for release of the DNA strands from RecA filaments. ATP binding is also required for RecA-driven branch migration, but non-hydrolyzable analogs of ATP can be substituted for ATP in this process, suggesting that nucleotide binding alone can provide conformational changes in RecA filaments that promote branch migration.

In addition to its role in homologous recombination, RecA functions as a coprotease for the LexA protein. In a healthy cell, LexA represses the expression of genes encoding DNA repair proteins (SOS genes). Upon injury of DNA, LexA catalyzes its own digestion, thereby allowing synthesis of necessary SOS proteins. However, LexA can only induce self-catalysis when activated by a ssDNA-RecA filament. A single filament will bind and activate several LexA proteins, each of which then cleaves other bound proteins. Thus, ssDNA-RecA, a product of DNA injury, stimulates DNA repair.



II. RecA: A Structural Overview

The RecA monomer consists of three domains, a large, central domain , surrounded by relatively small amino and carboxy domains. The central domain, involved in DNA and ATP binding, consists primarily of a twisted beta sheet with 8 b-strands , bounded by 8 a-helices . The amino domain contains a large a-helix and short b-strand, this a/b structure being important in formation of the RecA polymer . Three a-helices and a three-stranded b-sheet are found in the carboxy domain, which facilitates interfilament associations.



III. RecA Filament Structure

Catalysis of homologous recombination by RecA begins with the formation of a filament composed of RecA monomers around ssDNA. The RecA filament wraps around the DNA helically, with 6 monomers per revolution. The RecA helix is approximately 120 Å wide, with a central diameter of 25 Å. The carboxy termini of each monomer, which are believed to be important in interfilament interactions, project outward from the RecA helix. ATP is bound near the center of the helix. The amino domain of each RecA monomer is involved in maintaining the RecA polymer bonds. As described in the structural overview section, this region of the monomer contains a protruding a/ b unit .

The polymerization of RecA monomers into filaments involves extensive association of the amino domain of one monomer and the central domain of the next monomer in the filament (with a loss of 2,890 Å 2 of solvent-accessible surface area/monomer). This association can be visualized in a RecA dimer. Part of the subunit interface involves the packing of the amino a helix of one monomer between a complementary a helix and b sheet in the central domain of a neighboring monomer. Thus, the RecA filament has an amino domain-to-central domain polarity. The monomers are held together by a combination of hydrophobic and electrostatic interactions.

Experimental evidence supports the crystallographic data. Filament formation, for example, is severely inhibited among RecA monomers in which the amino terminal has been enzymatically removed. Similarly, proteins consisting only of the amino portion of RecA prevent polymerization via competitive inhibition of the central domain binding region. Mutation analyses have been used to identify residues at the subunit interface critical for RecA polymerization. Monomers in which lysine216
, phenylalanine217 , or arginine222 are replaced by other amino acids are unable to polymerize.



IV. DNA Binding Regions of RecA

The RecA monomer contains two DNA binding sites in the large central domain, one for binding ssDNA, and the other for binding duplex DNA. Both DNA binding regions include disordered loops (L1 & L2), containing residues with low electron density in the crystal. These loops, not shown in the structure, lie close to the filament axis, and therefore are juxtaposed with DNA. In the views that follow, the loops would project towards the viewer, i.e. towards the DNA in a RecA-DNA filament (see RecA Filament Structure, above).

The putative ssDNA binding region includes alpha helix G as well as L2 (not shown), between glu194 and thr210
. The putative binding site for duplex DNA is found in another disordered region, L1 (not shown), located between glu156 and gly165 .

Phylogenetic analyses have supported the conclusion that the regions containing L1 and L2 represent DNA binding regions. Because DNA binding is an essential function of RecA, the regions of the protein involved in this process should be highly conserved among bacterial species. Indeed, 10 of the 23 amino acids that compose the disordered loops are invariant in 16 different RecA proteins. Alpha helix G, located on the carboxy side of L2, is the most highly conserved region in the RecA monomer. At the boundary between alpha helix G and L2 are two invariant glycine residues, which, due to their small size, could allow maximal interaction between the negatively-charged sugar-phosphate backbone of the DNA molecule and the positively-charged amine groups of the helix .

Experimental studies have also confirmed the importance of the disordered regions in DNA binding. Mutations in several residues in and near the disordered loops leads to inhibition of DNA binding. In L2, these residues include glycine204, glutamate207, and glycine211. In L1, glycine160, glycine157, and arginine169 appear particularly important in binding duplex DNA. Furthermore, photocross-linking studies have mapped DNA binding to L1 and L2. Finally, a 20-amino acid peptide containing the L2 sequence is capable of independently binding DNA.

A study by Kumar and colleagues showed that binding of DNA to the RecA protein causes the disordered loops to assume alpha helical secondary structures. Interestingly, the amount of alpha helix induced by DNA binding is correlated with its base pair sequence. Less alpha helical structure is found in RecA proteins bound to CG-rich oligomers than to DNA fragments abundant in AT sequences. Furthermore, binding of homologous duplex DNA to ssDNA-RecA generates more alpha helix in the disordered loops than does binding of heterologous DNA. Thus, induction of alpha helix in the disordered loops may be a mechanism by which RecA pairs homologous strands.



V. The RecA ATP Binding Site

ATP binds to RecA in the central domain at a phosphate-binding loop (P-loop), a characteristic ATP binding region found in many proteins . Two amino acids in the P-loop, lysine72 and threonine73, are known to interact directly with the phosphate groups of the ATP . Like the DNA binding regions, the P-loop is located on the inner surface of the RecA filament. Bound ADP can be seen in this model of RecA. The a-carbons of lysine72 and threonine73 can be seen adjacent to the phosphates of the nucleotide.


VI. RecA Interfilament Interactions

The carboxy terminus of each RecA monomer functions in interfilament associations. In forming these interfilament bonds, the carboxy terminus of one monomer interacts with an area near the amino terminus of the neighboring filament . Obviously, these interfilament interactions are critical during the crystallization of the RecA protein for X-ray diffraction studies. However, associations between filaments may also be important biologically. In the Tif-1 mutation of RecA, glutamate38 is changed to lysine and isoleucine298 is converted to valine. Altering the RecA protein in this way prevents interfilament associations, and increases the efficiency of DNA binding. This observation suggests that RecA filament bundles form to prevent protein polymerization around incorrect targets (i.e. dsDNA, RNA), which would induce the SOS response unnecessarily.



VII. RecA Coprotease Activity

The LexA repressor is believed to bind to the monomer in a region on the carboxy side of the central domain
. Mutations in this region affect the ability of RecA to stimulate LexA autoproteolysis, but not homologous recombination catalyzation activity.



VIII. References

Story, R. M, I. T. Weber, and T. A. Steitz. 1992. The structure of the E. Coli RecA protein monomer and polymer. Nature 355: 318-325.

Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson. 1994. Molecular Biology of the Cell, 3rd edition. Garland Publishing, Inc: New York.

Konola, J. T., K. M. Logan, and K. L. Knight. 1994. Functional characterization of residues in the p-loop motif of the RecA protein ATP binding site. Journal of Molecular Biology 237: 20-34.

Kumar, K. A., S. Mahalakshmi, and K. Muniyappa. 1993. DNA-induced conformational changes in RecA protein. Journal of Biological Chemistry 268: 26162-26170.

Malkov, V. A. and R. D. Camerini-Otero. 1995. Photocross-links between single-stranded DNA and Escherichia coli RecA protein map to loops L1 (amino acid residues 157-164) and L2 (amino acid residues 195-209). Journal of Biological Chemistry 270: 30230-30233.

Mikawa, T., R. Masui, T. Ogawa, H. Ogawa, and S. Kuramitsu. 1995. N-terminal 33 amino acid residue of Escherichia coli RecA protein contributes to its self-assembly. Journal of Molecular Biology250: 471-483.

Skiba, M. C. and K. L. Knight. 1994. Functionally important residues at a subunit interface in the RecA protein from Escherichia coli. Journal of Biological Chemistry 269: 3823-3828.

Stryer, L. 1995. Biochemistry, 4th ed. W. H. Freeman and Company: New York.

Voloshin, O. N., L. Wang, and R. D. Camerini-Otero. 1996. Homologous DNA pairing promoted by a 20-amino acid peptide derived from RecA. Science 272: 868-872.


1, Kenyon College, Gambier, Ohio. A first draft of this exhibit was created for D. Marcey's Molecular Biology class, Biology 63.

2, Kenyon College, Gambier, Ohio. Present address: California Lutheran University. Address correspondence to this author (see below).

3, Kenyon College, Gambier, Ohio. This author transferred RasMol script files into the body of the exhibit text.


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