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Structural highlights
Function
RECC_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 almost certainly recognizes the wild-type Chi sequence, when added to isolated RecB increases its ATP-dependent helicase processivity. 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]
Publication Abstract from PubMed
In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 A resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.
Mechanism for nuclease regulation in RecBCD.,Wilkinson M, Chaban Y, Wigley DB Elife. 2016 Sep 20;5. pii: e18227. doi: 10.7554/eLife.18227. PMID:27644322[19]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ 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
- ↑ Arnold DA, Handa N, Kobayashi I, Kowalczykowski SC. A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi. J Mol Biol. 2000 Jul 14;300(3):469-79. PMID:10884344 doi:https://dx.doi.org/10.1006/jmbi.2000.3861
- ↑ 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
- ↑ Dillingham MS, Spies M, Kowalczykowski SC. RecBCD enzyme is a bipolar DNA helicase. Nature. 2003 Jun 19;423(6942):893-7. PMID:12815438 doi:https://dx.doi.org/10.1038/nature01673
- ↑ 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
- ↑ 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
- ↑ 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
- ↑ Wilkinson M, Chaban Y, Wigley DB. Mechanism for nuclease regulation in RecBCD. Elife. 2016 Sep 20;5. pii: e18227. doi: 10.7554/eLife.18227. PMID:27644322 doi:https://dx.doi.org/10.7554/eLife.18227
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