Biology:RecA

From HandWiki
recA bacterial DNA recombination protein
Crystal structure of a RecA (rainbow colored)-DNA (magenta) complex. PDB ID: 3cmt.[1]
Identifiers
SymbolRecA
PfamPF00154
Pfam clanCL0023
InterProIPR013765
PROSITEPDOC00131
SCOP22reb / SCOPe / SUPFAM
DNA recombination/repair protein RecA
Identifiers
OrganismEscherichia coli
SymbolrecA
Entrez947170
PDB3CMT (ECOD)
RefSeq (Prot)NP_417179.1
UniProtP0A7G6
Other data
EC number3.6.4.12
ChromosomeGenomic: 2.82 - 2.82 Mb

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: Biology: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. ↑ "Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures". Nature 453 (7194): 489–484. May 2008. doi:10.1038/nature06971. PMID 18497818. Bibcode: 2008Natur.453..489C. 
  2. ↑ "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. January 1980. doi:10.1073/pnas.77.1.313. PMID 6244554. Bibcode: 1980PNAS...77..313H. 
  3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 "RecA and DNA recombination: a review of molecular mechanisms". Biochemical Society Transactions 47 (5): 1511–1531. October 2019. doi:10.1042/BST20190558. PMID 31654073. https://hal.science/hal-02320683. 
  4. ↑ "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. July 2006. doi:10.1073/pnas.0604232103. PMID 16798872. Bibcode: 2006PNAS..10310328L. 
  5. ↑ "Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms". Journal of Molecular Evolution 44 (5): 528–541. May 1997. doi:10.1007/pl00006177. PMID 9115177. Bibcode: 1997JMolE..44..528B. 
  6. ↑ "Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein". Cell 69 (3): 457–470. May 1992. doi:10.1016/0092-8674(92)90447-k. PMID 1581961. Bibcode: 1992Cell...69..457S. 
  7. ↑ "RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange". Genes & Development 12 (9): 1248–1253. May 1998. doi:10.1101/gad.12.9.1248. PMID 9573041. 
  8. ↑ "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. December 1981. doi:10.1016/0092-8674(81)90393-7. PMID 6101204. 
  9. ↑ "Autodigestion of lexA and phage lambda repressors". Proceedings of the National Academy of Sciences of the United States of America 81 (5): 1375–1379. March 1984. doi:10.1073/pnas.81.5.1375. PMID 6231641. Bibcode: 1984PNAS...81.1375L. 
  10. ↑ 10.0 10.1 10.2 10.3 "RecA: Regulation and Mechanism of a Molecular Search Engine". Trends in Biochemical Sciences 41 (7): 491–507. July 2016. doi:10.1016/j.tibs.2016.04.002. PMID 27156412. 
  11. ↑ 11.0 11.1 "Allosteric movements in eubacterial RecA". Biophysical Reviews 4 (3): 199–208. October 2012. doi:10.1007/s12551-012-0097-4. PMID 28510010. 
  12. ↑ 12.0 12.1 12.2 Snyder & Champness molecular genetics of bacteria (Fifth ed.). Hoboken, NJ: Wiley. 2020. pp. 368–371. ISBN 978-1-55581-975-0. 
  13. ↑ 13.0 13.1 "All who wander are not lost: the search for homology during homologous recombination". Biochemical Society Transactions 52 (1): 367–377. February 2024. doi:10.1042/BST20230705. PMID 38323621. 
  14. ↑ "Homologous recombination and the repair of DNA double-strand breaks". The Journal of Biological Chemistry 293 (27): 10524–10535. July 2018. doi:10.1074/jbc.TM118.000372. PMID 29599286. 
  15. ↑ "RecA finds homologous DNA by reduced dimensionality search". Nature 597 (7876): 426–429. September 2021. doi:10.1038/s41586-021-03877-6. PMID 34471288. Bibcode: 2021Natur.597..426W. 
  16. ↑ "Break-ups and make-ups: DNA search and repair". Nature Reviews. Microbiology 20 (2): 66. February 2022. doi:10.1038/s41579-021-00671-z. PMID 34873308. 
  17. ↑ "How strand exchange protein function benefits from ATP hydrolysis". Current Opinion in Genetics & Development 71: 120–128. December 2021. doi:10.1016/j.gde.2021.06.016. PMID 34343922. 
  18. ↑ "RecA bundles mediate homology pairing between distant sisters during DNA break repair". Nature 506 (7487): 249–253. February 2014. doi:10.1038/nature12868. PMID 24362571. Bibcode: 2014Natur.506..249L. 
  19. ↑ Snyder & Champness molecular genetics of bacteria (Fifth ed.). Hoboken, NJ: Wiley. 2020. p. 259. ISBN 978-1-55581-975-0. 
  20. ↑ 20.0 20.1 "Targets for Combating the Evolution of Acquired Antibiotic Resistance". Biochemistry 54 (23): 3573–3582. June 2015. doi:10.1021/acs.biochem.5b00109. PMID 26016604. 
  21. ↑ "Targeting evolution to inhibit antibiotic resistance". The FEBS Journal 287 (20): 4341–4353. October 2020. doi:10.1111/febs.15370. PMID 32434280. 
  22. ↑ "Directed molecular screening for RecA ATPase inhibitors". Bioorganic & Medicinal Chemistry Letters 17 (12): 3249–3253. June 2007. doi:10.1016/j.bmcl.2007.04.013. PMID 17499507. 
  23. ↑ 23.0 23.1 23.2 "RecA: Regulation and Mechanism of a Molecular Search Engine". Trends in Biochemical Sciences 41 (6): 491–507. June 2016. doi:10.1016/j.tibs.2016.04.002. PMID 27156117. 
  24. ↑ "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. February 1965. doi:10.1073/pnas.53.2.451. PMID 14294081. Bibcode: 1965PNAS...53..451C. 
  25. ↑ 25.0 25.1 "recA mutants of E. coli K12: a personal turning point". BioEssays 18 (9): 767–772. September 1996. doi:10.1002/bies.950180912. PMID 8831293. 
  26. ↑ "The beginning of a genetic analysis of recombination proficiency". Journal of Cellular Physiology 70 (2). October 1967. doi:10.1002/jcp.1040700412. PMID 4867583. 
  27. ↑ "Do bacteria have sex?". Nature Reviews. Genetics 2 (8): 634–639. August 2001. doi:10.1038/35084593. PMID 11483988. 
  28. ↑ "The RecA protein: structure and function". Critical Reviews in Biochemistry and Molecular Biology 25 (6): 415–456. January 1990. doi:10.3109/10409239009090617. PMID 2292186. 
  29. ↑ "Specialized transduction of recA by bacteriophage lambda". Virology 70 (1): 221–222. March 1976. doi:10.1016/0042-6822(76)90258-0. PMID 769310. 
  30. ↑ "The structure of the E. coli recA protein monomer and polymer". Nature 355 (6358): 318–325. January 1992. doi:10.1038/355318a0. PMID 1731246. Bibcode: 1992Natur.355..318S. 
  31. ↑ "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. July 2001. doi:10.1073/pnas.131004998. PMID 11459950. Bibcode: 2001PNAS...98.8173C. 



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