5ld2

Structural highlights

Function

[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]} [RECD_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. In the holoenzyme this subunit contributes ssDNA-dependent ATPase and fast 5'-3' helicase activity. When added to pre-assembled RecBC greatly stimulates nuclease activity and augments holoenzyme processivity. Negatively regulates the RecA-loading ability of RecBCD.^{[23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]} [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.^{[37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54]}

References

1. ↑ 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
2. ↑ 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
3. ↑ 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
4. ↑ 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
5. ↑ 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:http://dx.doi.org/10.1038/nature01674
6. ↑ Dillingham MS, Spies M, Kowalczykowski SC. RecBCD enzyme is a bipolar DNA helicase. Nature. 2003 Jun 19;423(6942):893-7. PMID:12815438 doi:http://dx.doi.org/10.1038/nature01673
7. ↑ 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
8. ↑ 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:http://dx.doi.org/10.1074/jbc.M505520200
9. ↑ 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
10. ↑ 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:http://dx.doi.org/10.1021/bi051150v
11. ↑ 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:http://dx.doi.org/10.1101/gad.1605807
12. ↑ 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:http://dx.doi.org/10.1038/nsmb.1901
13. ↑ 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:http://dx.doi.org/10.1038/nature12333
14. ↑ 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:http://dx.doi.org/10.1016/j.jmb.2014.07.017
15. ↑ 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
16. ↑ 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
17. ↑ 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
18. ↑ 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
19. ↑ 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
20. ↑ 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
21. ↑ 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
22. ↑ 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:http://dx.doi.org/10.1006/jmbi.1998.2127
23. ↑ 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
24. ↑ Amundsen SK, Taylor AF, Smith GR. The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7399-404. PMID:10840065 doi:http://dx.doi.org/10.1073/pnas.130192397
25. ↑ 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:http://dx.doi.org/10.1038/nature01674
26. ↑ Dillingham MS, Spies M, Kowalczykowski SC. RecBCD enzyme is a bipolar DNA helicase. Nature. 2003 Jun 19;423(6942):893-7. PMID:12815438 doi:http://dx.doi.org/10.1038/nature01673
27. ↑ 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
28. ↑ 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:http://dx.doi.org/10.1074/jbc.M505520200
29. ↑ 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
30. ↑ 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:http://dx.doi.org/10.1101/gad.1605807
31. ↑ 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:http://dx.doi.org/10.1038/nature12333
32. ↑ 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
33. ↑ 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
34. ↑ 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
35. ↑ 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
36. ↑ 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:http://dx.doi.org/10.1006/jmbi.1998.2127
37. ↑ 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
38. ↑ 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:http://dx.doi.org/10.1006/jmbi.2000.3861
39. ↑ 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
40. ↑ 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:http://dx.doi.org/10.1038/nature01674
41. ↑ Dillingham MS, Spies M, Kowalczykowski SC. RecBCD enzyme is a bipolar DNA helicase. Nature. 2003 Jun 19;423(6942):893-7. PMID:12815438 doi:http://dx.doi.org/10.1038/nature01673
42. ↑ 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
43. ↑ 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:http://dx.doi.org/10.1074/jbc.M505520200
44. ↑ 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
45. ↑ 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:http://dx.doi.org/10.1038/nsmb.1901
46. ↑ 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:http://dx.doi.org/10.1038/nature12333
47. ↑ 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
48. ↑ 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
49. ↑ 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
50. ↑ 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
51. ↑ 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
52. ↑ 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
53. ↑ 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
54. ↑ 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:http://dx.doi.org/10.1006/jmbi.1998.2127
55. ↑ 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:http://dx.doi.org/10.7554/eLife.18227