Recombinase A: Difference between revisions

<Structure load='2REB' size='400' color='black' frame='true' align='right'/>

<scene name='41/413118/Reca_active_filament/1'>RecA active filament</scene>

<scene name='41/413118/Reca_adp_mg/3'>RecA with ADP and Mg bound</scene>

[[Recombinase A]] (RecA), a naturally aggregating protein  involved in DNA repair, is an important asset to the genetic integrity of the ''Escherichia coli'' (''E. coli'') genome.<ref name=Shan> Shan, Q.; Cox, M. M.; Inman, R. B. DNA Strand Exchange Promoted by RecA K72R. J. Biol. Chem. 1996, 271, 5712-5724. DOI:10.1074/jbc.271.10.5712 </ref> The survival of all species rely on such DNA repair processes. RecA homologues are found in all kingdoms including archaebacteria, eubacteria, and eukaryotes.<ref name=Brendel> Brendel, V.; Brocchieri, L.; Sandler, S.J.; Clark, A.J.; Karlin, S. Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms. J. Mol. Evol. 1997, 44, 528-541. DOI: 10.1007/PL00006177 </ref> Rad51, for example, is a RecA homologue found specifically in humans.<ref name=Baumann> Baumann, P.; Benson, F. E.; West, S. C. Human Rad51 Protein Promotes ATP-Dependent Homologous Pairing and Strand Transfer Reactions in Vitro. Cell. 1996, 87, 757-766. DOI: 10.1016/S0092-8674(00)81394-X </ref>  An over-expression of Rad51 in the nuclei of tumor cells when compared to those of normal breast tissue has been linked to sporadic, non-hereditary, breast cancers.<ref name=Maacke> Maacke, H.; Opitz, S.; Jost, K.; Hamdorf, W.; Henning, W. Krüger, S. Feller, A.C.; Lopens, A.; Diedrich, K.; Schwinger, E.; Stürzbecher, H.W. Over-expression of wild-type Rad51 correlates with histological grading of invasive ductal breast cancer. Int. J. Cancer. 2000, 88, 907-913. DOI: 10.1002/1097-0215(20001215)88:63.0.CO;2-4 </ref>

As RecA has many different functions, it also has several different binding sites for DNA, ATP, the LexA repressor, the λ repressor, as well as other RecA protein monomers to form a variety of oligomers.<ref name=Roca> Roca, A. I.; Cox, M. M. RecA Protein: Structure, Function, and Role in Recombinational DNA Repair. Prog. Nucleic Acid Res. Mol. Biol. 1997, 56, 129-223. DOI: 10.1016/S0079-6603(08)61005-3 </ref> <ref name=Story> Story, R. M.; Weber, I. T.; Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature (London) 1992, 355, 318-325. DOI: 10.1038/355318a0 </ref> <ref name=Walker> Walker, J. E.; Saraste, M.; Runswick, M. J. Gay, N. J. Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982, 1, 945-951. PMCID: PMC553140 </ref>  There are six RecA proteins per turn of the helix, and each individual monomer is capable of binding three base pairs of the extended conformation of DNA.<ref name=Cox> Cox, M. M. Motoring along with the bacterial RecA protein. Nat. Rev. Mol. Cell Biol. 2007, 8, 127-138. DOI: 10.1038/nrm2099 </ref>  This filament is not the only oligomer of RecA that exists in solution, however. Sattin and Goh have reported a variety of RecA structures in buffer, such as monomers, hexamers, rods/fibrils, protofibrils, and other small aggregates.<ref name=Sattin> Sattin, B. D.; Goh, M. C. Novel polymorphism of recA fibrils revealed by Atomic Force Microscopy. J. Biol. Phys. 2006, 32, 153-168. DOI: 10.1007/s10867-006-9010-3 </ref> Moreover, the type and amount of these different aggregation states is dynamic. Brenner and Zlotnick reported that the presence of monovalent salts changed the distribution of RecA aggregation states and that higher protein concentration tended to correspond to more aggregated structures.<ref name=Brenner> Brenner, S. L.; Zlotnick, A. RecA Protein Self-assembly: Multiple Discrete Aggregation States. J. Mol. Biol. 1988, 204, 959-972. DOI: 10.1016/0022-2836(88)90055-1 </ref> ATP hydrolysis occurs in the region of a loop consisting of amino acids 66-73 of the protein, which corresponds to the Walker A box motif and has the sequence GPESSGKT.<ref name=Roca> Roca, A. I.; Cox, M. M. RecA Protein: Structure, Function, and Role in Recombinational DNA Repair. Prog. Nucleic Acid Res. Mol. Biol. 1997, 56, 129-223. DOI: 10.1016/S0079-6603(08)61005-3 </ref> <ref name=Story> Story, R. M.; Weber, I. T.; Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature (London) 1992, 355, 318-325. DOI: 10.1038/355318a0 </ref> This sequence corresponds to a variation known as the phosphate binding loop, which has a sequence [G/A]XXXXGK[T/S] found in many nucleoside triphosphate (NTP)-binding proteins.<ref name=Story> Story, R. M.; Weber, I. T.; Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature (London) 1992, 355, 318-325. DOI: 10.1038/355318a0 </ref> <ref name=Walker> Walker, J. E.; Saraste, M.; Runswick, M. J. Gay, N. J. Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982, 1, 945-951. PMCID: PMC553140 </ref> Several of the residues in this phosphate binding loop can be seen interacting with the β and γ phosphates of ATP in the ATP-binding site proposed by Story and Steitz.<ref name=Story> Story, R. M.; Weber, I. T.; Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature (London) 1992, 355, 318-325. DOI: 10.1038/355318a0 </ref> The binding of various ligands to RecA has been shown to change the pitch, the “distance covered by each turn of the helix,” of the protein filament.<ref name=Ellouze> Ellouze, C.; Takahashi, M.; Wittung, P.; Mortensen, K.; Schnarr, M.; Nordén, B. Evidence for elongation of helical pitch of the helical pitch of the RecA filament upon ATP and ADP binding using small-angle neutron scattering. Eur. J. Biochem. 1995,233, 579-583. DOI: 10.1111/j.1432-1033.1995.579_2.x </ref> <ref name=Menetski> Menetski, J. P.; Kowalczykowski, S. C. Interaction of recA protein with single-stranded DNA: Quantitative aspects of binding affinity modulation by nucleotide cofactors. J. Mol. Biol. 1985, 181, 281-295. DOI: 10.1016/0022-2836(85)90092-0 </ref> RecA in the absence of any cofactor is in a “closed” conformation with a helical pitch of 7 nm (DNA binding to the RecA does not alter the pitch significantly). RecA bound to ATP increases the pitch to 9 nm.<ref name=Ellouze> Ellouze, C.; Takahashi, M.; Wittung, P.; Mortensen, K.; Schnarr, M.; Nordén, B. Evidence for elongation of helical pitch of the helical pitch of the RecA filament upon ATP and ADP binding using small-angle neutron scattering. Eur. J. Biochem. 1995,233, 579-583. DOI: 10.1111/j.1432-1033.1995.579_2.x </ref> This RecA-ATP structure is marked by a higher affinity for DNA.<ref name=Roca> Roca, A. I.; Cox, M. M. RecA Protein: Structure, Function, and Role in Recombinational DNA Repair. Prog. Nucleic Acid Res. Mol. Biol. 1997, 56, 129-223. DOI: 10.1016/S0079-6603(08)61005-3 </ref> <ref name=Menetski> Menetski, J. P.; Kowalczykowski, S. C. Interaction of recA protein with single-stranded DNA: Quantitative aspects of binding affinity modulation by nucleotide cofactors. J. Mol. Biol. 1985, 181, 281-295. DOI: 10.1016/0022-2836(85)90092-0 </ref> However, the binding of ADP to RecA only raises the pitch to 8.2 nm,<ref name=Ellouze> Ellouze, C.; Takahashi, M.; Wittung, P.; Mortensen, K.; Schnarr, M.; Nordén, B. Evidence for elongation of helical pitch of the helical pitch of the RecA filament upon ATP and ADP binding using small-angle neutron scattering. Eur. J. Biochem. 1995,233, 579-583. DOI: 10.1111/j.1432-1033.1995.579_2.x </ref> the conformation of which is known to have a lower affinity for DNA.<ref name=Roca> Roca, A. I.; Cox, M. M. RecA Protein: Structure, Function, and Role in Recombinational DNA Repair. Prog. Nucleic Acid Res. Mol. Biol. 1997, 56, 129-223. DOI: 10.1016/S0079-6603(08)61005-3 </ref> <ref name=Menetski> Menetski, J. P.; Kowalczykowski, S. C. Interaction of recA protein with single-stranded DNA: Quantitative aspects of binding affinity modulation by nucleotide cofactors. J. Mol. Biol. 1985, 181, 281-295. DOI: 10.1016/0022-2836(85)90092-0 </ref>  

High salt concentrations have also been shown to be able to elongate the RecA protein filament as well. Petukhov et al. demonstrated that a high concentration of NaCl increased the helical pitch from 7.8 to 8.6 nm.<ref name=Peukhov> Peukhov, M.; Lebedev, D.; Shalguev, V.; Islamov, A.; Kruklin, A.; Lanzov, V.; Isaev-Ivanov, V. Conformational Flexibility of RecA Protein Filament: Transitions between Compressed and Stretched States. Proteins: Struct.,Funct., Bioinf. 2006,65, 296-304. DOI: 10.1002/prot.21116 </ref> Thus, high salt concentrations appear to induce the active (stretched) form of RecA in the absence of DNA.<ref name=Peukhov> Peukhov, M.; Lebedev, D.; Shalguev, V.; Islamov, A.; Kruklin, A.; Lanzov, V.; Isaev-Ivanov, V. Conformational Flexibility of RecA Protein Filament: Transitions between Compressed and Stretched States. Proteins: Struct.,Funct., Bioinf. 2006,65, 296-304. DOI: 10.1002/prot.21116 </ref>  Other studies have found that the free Magnesium ion binds to RecA and extends the filament more than 150% compared to the filament when DNA is bound.<ref name=Lusetti>  Lusetti, S. L.; Shaw, J. J.; Cox, M. M. Magnesium Ion-dependent Activation of the RecA Protein