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RecA is a 38 kilodalton protein essential for the repair and maintenance of DNA in bacteria.[2] It functions as a recombinase and strand-exchange protein, catalyzing the central steps of homologous recombination by forming nucleoprotein filaments on single-stranded DNA.[3] Structural and functional homologs to RecA have been found in all kingdoms of life.[4][5] RecA serves as an archetype for this class of homologous DNA repair proteins. The homologous protein is called RAD51 in eukaryotes and RadA in archaea.[6][7]

RecA has multiple activities, all related to DNA repair. As a recombinase, it mediates ATP-dependent strand exchange between homologous DNA molecules, driving the key pairing and heteroduplex formation steps of recombinational repair.[3] In the bacterial SOS response, it functions as a co-protease[8] in the autocatalytic cleavage of the LexA repressor and the λ repressor.[9]

Structure

The E. coli RecA monomer (352 amino acids, ~37.8 kDa) is organized into three structural domains:

  • small N-terminal domain (NTD, residues ~1–33) The NTD mediates monomer–monomer interactions during filament polymerization and additionally facilitates presynaptic filament formation and dsDNA capture, functions that are evolutionarily conserved across the RecA/RAD51/RadA family.[10]
  • central core ATPase domain (CAD, residues ~34–240) The CAD constitutes the functional heart of the protein, housing two Walker motifs (Walker A (P-loop) and Walker B) responsible for ATP binding and hydrolysis, as well as the DNA-binding loops L1 and L2 that contact single-stranded DNA within the filament.[3]
  • large C-terminal domain (CTD, residues ~241–352).[10] The CTD contributes to secondary DNA binding (the interaction with the incoming duplex during homology search) and contains a second nucleotide-binding site implicated in allosteric regulation of filament activity.[11]

RecA monomers polymerize cooperatively onto ssDNA in the presence of ATP to form a right-handed helical nucleoprotein filament with approximately 6 monomers per turn and a helical pitch of ~95 Å, in which the DNA is stretched ~1.5-fold relative to B-form and held in a conformation competent for homology search and strand exchange.[3][10] The filament exists in two conformational states — an extended, ATP-bound active form and a compressed, ADP-bound inactive form — with cooperative transitions between neighboring monomers ensuring that the filament remains catalytically competent throughout the ATPase cycle.[11][10]

Function

Homologous recombination

The RecA protein binds strongly and in long clusters to ssDNA to form a nucleoprotein filament.[12] This is also called a presynaptic filament.[3] The presynaptic filament has an inactive and active conformation. RecA must be bound to ATP to form an active filament. The activated filament searches for a homologous region of dsDNA to bind to, a process known as synapsis.

The mechanisms of the RecA homology search are not fully understood.[12][13] The RecA filament searches the dsDNA in 8 base pair segments.[14] When the threshold of 8-bases of homology is exceeded, the filament complex is stabilized.[3] In 2021, Witkor et al., demonstrated that the RecA filament uses a “reduced dimensionality” search mechanism.[15][16]

Once the filament has located and bound to a complementary sequence of dsDNA, strand exchange occurs.[12] This reaction occurs in the 5′ to 3′ direction.[13]

Since it is a DNA-dependent ATPase, RecA contains an additional site for binding and hydrolyzing ATP. RecA associates more tightly with DNA when it has ATP bound than when it has ADP bound.[17]

Homologous recombination events mediated by RecA can occur in Escherichia coli during the period after DNA replication when sister loci remain close. RecA can also mediate homology pairing, homologous recombination, and DNA break repair between distant sister loci that had segregated to opposite halves of the E. coli cell.[18]

Natural transformation

Main article: Natural competence

Natural bacterial transformation involves the transfer of DNA from one bacterium to another (ordinarily of the same species) and the integration of the donor DNA into the recipient chromosome by homologous recombination, a process mediated by the RecA protein. In some bacteria, the recA gene is induced in response to the bacterium becoming competent, the physiological state required for transformation.[19]

Clinical significance

RecA has been proposed as a potential drug target for bacterial infections.[20] Small molecules that interfere with RecA function have been identified.[21][22] Since many antibiotics lead to DNA damage, and all bacteria rely on RecA to fix this damage, inhibitors of RecA could be used to enhance the toxicity of antibiotics. Inhibitors of RecA may also delay or prevent the appearance of bacterial drug resistance.[20]

History

RecA was discovered in 1965 by Alvin J. Clark and Ann Dee Margulies in genetic screens for recombination deficient strains of E. coli.[23][24] The gene name “rec”, first published in 1969, was chosen to indicate its involvement in recombination.[25][26][27] In 1976, the recA gene was cloned for the first time by Kevin McEntee.[28][29] Shortly after, the protein was purified for the first time by several groups.[25][23] Purification of the protein led to a number of breakthroughs on the biochemical properties of RecA. The first crystal structure of RecA was published in 1992, nearly 30 years after the protein was discovered.[30]

Later research identified related proteins, including RecBCD and RecF.[23][31]

References

  1. ^ Chen Z, Yang H, Pavletich NP (May 2008). “Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures”. Nature. 453 (7194): 489–484. Bibcode:2008Natur.453..489C. doi:10.1038/nature06971. PMID 18497818. S2CID 4416531.
  2. ^ Horii T, Ogawa T, Ogawa H (January 1980). “Organization of the recA gene of Escherichia coli”. Proceedings of the National Academy of Sciences of the United States of America. 77 (1): 313–317. Bibcode:1980PNAS…77..313H. doi:10.1073/pnas.77.1.313. PMC 348260. PMID 6244554.
  3. ^ a b c d e f Del Val E, Nasser W, Abaibou H, Reverchon S (October 2019). “RecA and DNA recombination: a review of molecular mechanisms”. Biochemical Society Transactions. 47 (5): 1511–1531. doi:10.1042/BST20190558. PMID 31654073.
  4. ^ Lin Z, Kong H, Nei M, Ma H (July 2006). “Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer”. Proceedings of the National Academy of Sciences of the United States of America. 103 (27): 10328–10333. Bibcode:2006PNAS..10310328L. doi:10.1073/pnas.0604232103. PMC 1502457. PMID 16798872.
  5. ^ Brendel V, Brocchieri L, Sandler SJ, Clark AJ, Karlin S (May 1997). “Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms”. Journal of Molecular Evolution. 44 (5): 528–541. Bibcode:1997JMolE..44..528B. doi:10.1007/pl00006177. PMID 9115177.
  6. ^ Shinohara A, Ogawa H, Ogawa T (May 1992). “Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein”. Cell. 69 (3): 457–470. Bibcode:1992Cell…69..457S. doi:10.1016/0092-8674(92)90447-k. PMID 1581961. S2CID 35937283.
  7. ^ Seitz EM, Brockman JP, Sandler SJ, Clark AJ, Kowalczykowski SC (May 1998). “RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange”. Genes & Development. 12 (9): 1248–1253. doi:10.1101/gad.12.9.1248. PMC 316774. PMID 9573041.
  8. ^ Horii T, Ogawa T, Nakatani T, Hase T, Matsubara H, Ogawa H (December 1981). “Regulation of SOS functions: purification of E. coli LexA protein and determination of its specific site cleaved by the RecA protein”. Cell. 27 (3 Pt 2): 515–522. doi:10.1016/0092-8674(81)90393-7. PMID 6101204. S2CID 45482725.
  9. ^ Little JW (March 1984). “Autodigestion of lexA and phage lambda repressors”. Proceedings of the National Academy of Sciences of the United States of America. 81 (5): 1375–1379. Bibcode:1984PNAS…81.1375L. doi:10.1073/pnas.81.5.1375. PMC 344836. PMID 6231641.
  10. ^ a b c d Bell JC, Kowalczykowski SC (July 2016). “RecA: Regulation and Mechanism of a Molecular Search Engine”. Trends in Biochemical Sciences. 41 (7): 491–507. doi:10.1016/j.tibs.2016.04.002. PMC 4892382. PMID 27156412.
  11. ^ a b Chandran A, Vijayan M (October 2012). “Allosteric movements in eubacterial RecA”. Biophysical Reviews. 4 (3): 199–208. doi:10.1007/s12551-012-0097-4. PMC 5418409. PMID 28510010.
  12. ^ a b c Henkin TM, Peters JE, Snyder L, Champness W (2020). Snyder & Champness molecular genetics of bacteria (Fifth ed.). Hoboken, NJ: Wiley. pp. 368–371. ISBN 978-1-55581-975-0.
  13. ^ a b Hu J, Crickard JB (February 2024). “All who wander are not lost: the search for homology during homologous recombination”. Biochemical Society Transactions. 52 (1): 367–377. doi:10.1042/BST20230705. PMC 10903458. PMID 38323621.
  14. ^ Wright WD, Shah SS, Heyer WD (July 2018). “Homologous recombination and the repair of DNA double-strand breaks”. The Journal of Biological Chemistry. 293 (27): 10524–10535. doi:10.1074/jbc.TM118.000372. PMC 6036207. PMID 29599286.
  15. ^ Wiktor J, Gynnå AH, Leroy P, Larsson J, Coceano G, Testa I, et al. (September 2021). “RecA finds homologous DNA by reduced dimensionality search”. Nature. 597 (7876): 426–429. Bibcode:2021Natur.597..426W. doi:10.1038/s41586-021-03877-6. PMC 8443446. PMID 34471288.
  16. ^ Lalande E, El Sayyed H (February 2022). “Break-ups and make-ups: DNA search and repair”. Nature Reviews. Microbiology. 20 (2): 66. doi:10.1038/s41579-021-00671-z. PMID 34873308.
  17. ^ Reitz D, Chan YL, Bishop DK (December 2021). “How strand exchange protein function benefits from ATP hydrolysis”. Current Opinion in Genetics & Development. 71: 120–128. doi:10.1016/j.gde.2021.06.016. PMC 8671154. PMID 34343922.
  18. ^ Lesterlin C, Ball G, Schermelleh L, Sherratt DJ (February 2014). “RecA bundles mediate homology pairing between distant sisters during DNA break repair”. Nature. 506 (7487): 249–253. Bibcode:2014Natur.506..249L. doi:10.1038/nature12868. PMC 3925069. PMID 24362571.
  19. ^ Henkin TM, Peters JE, Snyder L, Champness W (2020). Snyder & Champness molecular genetics of bacteria (Fifth ed.). Hoboken, NJ: Wiley. p. 259. ISBN 978-1-55581-975-0.
  20. ^ a b Culyba MJ, Mo CY, Kohli RM (June 2015). “Targets for Combating the Evolution of Acquired Antibiotic Resistance”. Biochemistry. 54 (23): 3573–3582. doi:10.1021/acs.biochem.5b00109. PMC 4471857. PMID 26016604.
  21. ^ Merrikh H, Kohli RM (October 2020). “Targeting evolution to inhibit antibiotic resistance”. The FEBS Journal. 287 (20): 4341–4353. doi:10.1111/febs.15370. PMC 7578009. PMID 32434280.
  22. ^ Wigle TJ, Singleton SF (June 2007). “Directed molecular screening for RecA ATPase inhibitors”. Bioorganic & Medicinal Chemistry Letters. 17 (12): 3249–3253. doi:10.1016/j.bmcl.2007.04.013. PMC 1933586. PMID 17499507.
  23. ^ a b c Bell JC, Kowalczykowski SC (June 2016). “RecA: Regulation and Mechanism of a Molecular Search Engine”. Trends in Biochemical Sciences. 41 (6): 491–507. doi:10.1016/j.tibs.2016.04.002. PMC 4892382. PMID 27156117.
  24. ^ Clark AJ, Margulies AD (February 1965). “Isolation and characterization of recombination-deficient mutants of Escherichia coli K12″. Proceedings of the National Academy of Sciences of the United States of America. 53 (2): 451–459. Bibcode:1965PNAS…53..451C. doi:10.1073/pnas.53.2.451. PMC 219534. PMID 14294081.
  25. ^ a b Clark AJ (September 1996). “recA mutants of E. coli K12: a personal turning point”. BioEssays. 18 (9): 767–772. doi:10.1002/bies.950180912. PMID 8831293.
  26. ^ Clark AJ (October 1967). “The beginning of a genetic analysis of recombination proficiency”. Journal of Cellular Physiology. 70 (2) Suppl:165–80. doi:10.1002/jcp.1040700412. PMID 4867583.
  27. ^ Redfield RJ (August 2001). “Do bacteria have sex?”. Nature Reviews. Genetics. 2 (8): 634–639. doi:10.1038/35084593. PMID 11483988.
  28. ^ Roca AI, Cox MM (January 1990). “The RecA protein: structure and function”. Critical Reviews in Biochemistry and Molecular Biology. 25 (6): 415–456. doi:10.3109/10409239009090617. PMID 2292186.
  29. ^ McEntee K (March 1976). “Specialized transduction of recA by bacteriophage lambda”. Virology. 70 (1): 221–222. doi:10.1016/0042-6822(76)90258-0. PMID 769310.
  30. ^ Story RM, Weber IT, Steitz TA (January 1992). “The structure of the E. coli recA protein monomer and polymer”. Nature. 355 (6358): 318–325. Bibcode:1992Natur.355..318S. doi:10.1038/355318a0. PMID 1731246.
  31. ^ Cox MM (July 2001). “Historical overview: searching for replication help in all of the rec places”. Proceedings of the National Academy of Sciences of the United States of America. 98 (15): 8173–8180. Bibcode:2001PNAS…98.8173C. doi:10.1073/pnas.131004998. PMC 37418. PMID 11459950.