RECB_ECOLI A helicase/nuclease that prepares dsDNA breaks (DSB) for recombinational DNA repair. Binds to DSBs and unwinds DNA via a rapid (>1 kb/second) and highly processive (>30 kb) ATP-dependent bidirectional helicase. Unwinds dsDNA until it encounters a Chi (crossover hotspot instigator, 5'-GCTGGTGG-3') sequence from the 3' direction. Cuts ssDNA a few nucleotides 3' to Chi site, by nicking one strand or switching the strand degraded (depending on the reaction conditions). The properties and activities of the enzyme are changed at Chi. The Chi-altered holoenzyme produces a long 3'-ssDNA overhang which facilitates RecA-binding to the ssDNA for homologous DNA recombination and repair. Holoenzyme degrades any linearized DNA that is unable to undergo homologous recombination (PubMed:4562392, PubMed:4552016, PubMed:123277). In the holoenzyme this subunit contributes ATPase, 3'-5' helicase, exonuclease activity and loads RecA onto ssDNA. The RecBC complex requires the RecD subunit for nuclease activity, but can translocate along ssDNA in both directions.[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]
Evolutionary Conservation
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Publication Abstract from PubMed
RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.
Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks.,Singleton MR, Dillingham MS, Gaudier M, Kowalczykowski SC, Wigley DB Nature. 2004 Nov 11;432(7014):187-93. PMID:15538360[23]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
↑Taylor AF, Smith GR. Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits. Genes Dev. 1999 Apr 1;13(7):890-900. PMID:10197988
↑Zhang XJ, Julin DA. Isolation and characterization of the C-terminal nuclease domain from the RecB protein of Escherichia coli. Nucleic Acids Res. 1999 Nov 1;27(21):4200-7. PMID:10518611
↑Arnold DA, Kowalczykowski SC. Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme. J Biol Chem. 2000 Apr 21;275(16):12261-5. PMID:10766864
↑Benzinger R, Enquist LW, Skalka A. Transfection of Escherichia coli spheroplasts. V. Activity of recBC nuclease in rec+ and rec minus spheroplasts measured with different forms of bacteriophage DNA. J Virol. 1975 Apr;15(4):861-71. PMID:123277
↑Taylor AF, Smith GR. RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity. Nature. 2003 Jun 19;423(6942):889-93. PMID:12815437 doi:https://dx.doi.org/10.1038/nature01674
↑Taylor AF, Smith GR. RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5226-30. PMID:1535156
↑Dillingham MS, Webb MR, Kowalczykowski SC. Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme. J Biol Chem. 2005 Nov 4;280(44):37069-77. Epub 2005 Jul 22. PMID:16041061 doi:https://dx.doi.org/10.1074/jbc.M505520200
↑Masterson C, Boehmer PE, McDonald F, Chaudhuri S, Hickson ID, Emmerson PT. Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits. J Biol Chem. 1992 Jul 5;267(19):13564-72. PMID:1618858
↑Sun JZ, Julin DA, Hu JS. The nuclease domain of the Escherichia coli RecBCD enzyme catalyzes degradation of linear and circular single-stranded and double-stranded DNA. Biochemistry. 2006 Jan 10;45(1):131-40. PMID:16388588 doi:https://dx.doi.org/10.1021/bi051150v
↑Amundsen SK, Taylor AF, Reddy M, Smith GR. Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots. Genes Dev. 2007 Dec 15;21(24):3296-307. PMID:18079176 doi:https://dx.doi.org/10.1101/gad.1605807
↑Wu CG, Bradford C, Lohman TM. Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor. Nat Struct Mol Biol. 2010 Oct;17(10):1210-7. doi: 10.1038/nsmb.1901. Epub 2010, Sep 19. PMID:20852646 doi:https://dx.doi.org/10.1038/nsmb.1901
↑Liu B, Baskin RJ, Kowalczykowski SC. DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate. Nature. 2013 Aug 22;500(7463):482-5. doi: 10.1038/nature12333. Epub 2013 Jul 14. PMID:23851395 doi:https://dx.doi.org/10.1038/nature12333
↑Taylor AF, Amundsen SK, Guttman M, Lee KK, Luo J, Ranish J, Smith GR. Control of RecBCD enzyme activity by DNA binding- and Chi hotspot-dependent conformational changes. J Mol Biol. 2014 Oct 23;426(21):3479-99. doi: 10.1016/j.jmb.2014.07.017. Epub, 2014 Jul 27. PMID:25073102 doi:https://dx.doi.org/10.1016/j.jmb.2014.07.017
↑Karu AE, MacKay V, Goldmark PJ, Linn S. The recBC deoxyribonuclease of Escherichia coli K-12. Substrate specificity and reaction intermediates. J Biol Chem. 1973 Jul 25;248(14):4874-84. PMID:4268693
↑Goldmark PJ, Linn S. Purification and properties of the recBC DNase of Escherichia coli K-12. J Biol Chem. 1972 Mar 25;247(6):1849-60. PMID:4552016
↑Simmon VF, Lederberg S. Degradation of bacteriophage lambda deoxyribonucleic acid after restriction by Escherichia coli K-12. J Bacteriol. 1972 Oct;112(1):161-9. PMID:4562392
↑Dixon DA, Kowalczykowski SC. Role of the Escherichia coli recombination hotspot, chi, in RecABCD-dependent homologous pairing. J Biol Chem. 1995 Jul 7;270(27):16360-70. PMID:7608206
↑Bianco PR, Kowalczykowski SC. The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'. Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6706-11. PMID:9192629
↑Anderson DG, Kowalczykowski SC. The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner. Cell. 1997 Jul 11;90(1):77-86. PMID:9230304
↑Yu M, Souaya J, Julin DA. The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):981-6. PMID:9448271
↑Yu M, Souaya J, Julin DA. Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme. J Mol Biol. 1998 Nov 6;283(4):797-808. PMID:9790841 doi:https://dx.doi.org/10.1006/jmbi.1998.2127
↑Singleton MR, Dillingham MS, Gaudier M, Kowalczykowski SC, Wigley DB. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature. 2004 Nov 11;432(7014):187-93. PMID:15538360 doi:10.1038/nature02988
