Reconstruction of Bacterial Transcription-coupled Repair at Single-molecule Resolution

Overview

Journal Nature

Specialty Science

Date 2016 Aug 4

PMID 27487215

Citations 45

Authors

Jun Fan

Mathieu Leroux-Coyau

Nigel J Savery

Terence R Strick

Affiliations

Soon will be listed here.

Abstract

Escherichia coli Mfd translocase enables transcription-coupled repair by displacing RNA polymerase (RNAP) stalled on a DNA lesion and then coordinating assembly of the UvrAB(C) components at the damage site. Recent studies have shown that after binding to and dislodging stalled RNAP, Mfd remains on the DNA in the form of a stable, slowly translocating complex with evicted RNAP attached. Here we find, using a series of single-molecule assays, that recruitment of UvrA and UvrAB to Mfd-RNAP arrests the translocating complex and causes its dissolution. Correlative single-molecule nanomanipulation and fluorescence measurements show that dissolution of the complex leads to loss of both RNAP and Mfd. Subsequent DNA incision by UvrC is faster than when only UvrAB(C) are available, in part because UvrAB binds 20-200 times more strongly to Mfd–RNAP than to DNA damage. These observations provide a quantitative framework for comparing complementary DNA repair pathways in vivo.

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References

Revyakin A, Ebright R, Strick T . Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments. Nat Methods. 2005; 2(2):127-38. DOI: 10.1038/nmeth0205-127. View

Smith A, Savery N . RNA polymerase mutants defective in the initiation of transcription-coupled DNA repair. Nucleic Acids Res. 2005; 33(2):755-64. PMC: 548365. DOI: 10.1093/nar/gki225. View

Smith A, Szczelkun M, Savery N . Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase. Nucleic Acids Res. 2007; 35(6):1802-11. PMC: 1874598. DOI: 10.1093/nar/gkm019. View

Mellon I, Spivak G, HANAWALT P . Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell. 1987; 51(2):241-9. DOI: 10.1016/0092-8674(87)90151-6. View

WITKIN E . Radiation-induced mutations and their repair. Science. 1966; 152(3727):1345-53. DOI: 10.1126/science.152.3727.1345. View

Savery N . The molecular mechanism of transcription-coupled DNA repair. Trends Microbiol. 2007; 15(7):326-33. DOI: 10.1016/j.tim.2007.05.005. View

Verhoeven E, Wyman C, Moolenaar G, Hoeijmakers J, Goosen N . Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition. EMBO J. 2001; 20(3):601-11. PMC: 133479. DOI: 10.1093/emboj/20.3.601. View

Revyakin A, Ebright R, Strick T . Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation. Proc Natl Acad Sci U S A. 2004; 101(14):4776-80. PMC: 387324. DOI: 10.1073/pnas.0307241101. View

Deaconescu A, Chambers A, Smith A, Nickels B, Hochschild A, Savery N . Structural basis for bacterial transcription-coupled DNA repair. Cell. 2006; 124(3):507-20. DOI: 10.1016/j.cell.2005.11.045. View

10.

Revyakin A, Allemand J, Croquette V, Ebright R, Strick T . Single-molecule DNA nanomanipulation: detection of promoter-unwinding events by RNA polymerase. Methods Enzymol. 2004; 370:577-98. DOI: 10.1016/S0076-6879(03)70049-4. View

11.

Srivastava D, Darst S . Derepression of bacterial transcription-repair coupling factor is associated with a profound conformational change. J Mol Biol. 2010; 406(2):275-84. PMC: 3031748. DOI: 10.1016/j.jmb.2010.12.004. View

12.

Manelyte L, Kim Y, Smith A, Smith R, Savery N . Regulation and rate enhancement during transcription-coupled DNA repair. Mol Cell. 2010; 40(5):714-24. PMC: 3025350. DOI: 10.1016/j.molcel.2010.11.012. View

13.

Selby C, Sancar A . Structure and function of the (A)BC excinuclease of Escherichia coli. Mutat Res. 1990; 236(2-3):203-11. DOI: 10.1016/0921-8777(90)90005-p. View

14.

Howan K, Smith A, Westblade L, Joly N, Grange W, Zorman S . Initiation of transcription-coupled repair characterized at single-molecule resolution. Nature. 2012; 490(7420):431-4. PMC: 3475728. DOI: 10.1038/nature11430. View

15.

van den Broek B, Noom M, Wuite G . DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway. Nucleic Acids Res. 2005; 33(8):2676-84. PMC: 1092278. DOI: 10.1093/nar/gki565. View

16.

Wang M, Schnitzer M, Yin H, Landick R, Gelles J, Block S . Force and velocity measured for single molecules of RNA polymerase. Science. 1998; 282(5390):902-7. DOI: 10.1126/science.282.5390.902. View

17.

Duboc C, Graves E, Strick T . Simple calibration of TIR field depth using the supercoiling response of DNA. Methods. 2016; 105:56-61. DOI: 10.1016/j.ymeth.2016.03.028. View

18.

Strick T, Allemand J, Bensimon D, Bensimon A, Croquette V . The elasticity of a single supercoiled DNA molecule. Science. 1996; 271(5257):1835-7. DOI: 10.1126/science.271.5257.1835. View

19.

Graves E, Duboc C, Fan J, Stransky F, Leroux-Coyau M, Strick T . A dynamic DNA-repair complex observed by correlative single-molecule nanomanipulation and fluorescence. Nat Struct Mol Biol. 2015; 22(6):452-7. DOI: 10.1038/nsmb.3019. View

20.

Haines N, Kim Y, Smith A, Savery N . Stalled transcription complexes promote DNA repair at a distance. Proc Natl Acad Sci U S A. 2014; 111(11):4037-42. PMC: 3964087. DOI: 10.1073/pnas.1322350111. View