Investigation of Intradomain Motions of a Y-Family DNA Polymerase during Substrate Binding and Catalysis.
DNA polymerases catalyze DNA synthesis through a stepwise kinetic mechanism that begins with binding to DNA, followed by selection, binding, and incorporation of a nucleotide into an elongating primer. It is hypothesized that subtle active site adjustments in a polymerase to align reactive moieties limit the rate of correct nucleotide incorporation. DNA damage can impede this process for many DNA polymerases, causing replication fork stalling, genetic mutations, and potentially cell death. However, specialized Y-family DNA polymerases are structurally evolved to efficiently bypass DNA damage in vivo, albeit at the expense of replication fidelity. Dpo4, a model Y-family polymerase from Sulfolobus solfataricus, has been well-studied kinetically, structurally, and computationally, which yielded a mechanistic understanding of how the Y-family DNA polymerases achieve their unique catalytic properties. We previously employed a real-time Förster resonance energy transfer (FRET) technique to characterize the global conformational motions of Dpo4 during DNA binding as well as nucleotide binding and incorporation by monitoring changes in distance between sites on the polymerase and DNA, and even between domains of Dpo4. Here, we extend the utility of our FRET methodology to observe conformational transitions within individual domains of Dpo4 during DNA binding and nucleotide incorporation. The results of this novel, intradomain FRET approach unify findings from many studies to fully clarify the complex DNA binding mechanism of Dpo4. Furthermore, intradomain motions in the Finger domain during nucleotide binding and incorporation, for the first time, report on the rate-limiting step of a single-nucleotide addition catalyzed by Dpo4.
Department of Chemistry and Biochemistry, The Ohio State University , Columbus, Ohio 43210, United States.|Ohio State Biochemistry Program, The Ohio State University , Columbus, Ohio 43210, United States.
Austin T
AT
Raper
Department of Chemistry and Biochemistry, The Ohio State University , Columbus, Ohio 43210, United States.|Ohio State Biochemistry Program, The Ohio State University , Columbus, Ohio 43210, United States.
Zucai
Z
Suo
D002384:Catalysis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D011487:Protein Conformation; D013379:Substrate Specificity
D004259:DNA-Directed DNA Polymerase
Biochemistry. 1995 Dec 12;34(49):15881-5
8519743
Nucleic Acids Res. 2008 Apr;36(6):1990-2001
18276639
Nucleic Acids Res. 2014 Feb;42(4):2555-63
24270793
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8868-73
10908673
Proteins. 2001 Sep 1;44(4):418-27
11484219
J Biol Chem. 2004 Jul 30;279(31):32932-40
15155753
Mol Cell. 2001 Jul;8(1):7-8
11515498
Biomol NMR Assign. 2011 Oct;5(2):195-8
21337030
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8368-75
11459977
Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13737-41
19666553
Methods Enzymol. 1992;211:353-88
1406315
J Mol Biol. 2014 Aug 12;426(16):2901-2917
24931550
Proc Natl Acad Sci U S A. 2009 Nov 17;106(46):19346-51
19887638
Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11182-7
18678900
Biochemistry. 2014 May 6;53(17):2804-14
24716482
J Nucleic Acids. 2010 Aug 22;2010:
20847947
Biochemistry. 2016 Apr 12;55(14):2187-96
27002236
Biochemistry. 2012 Apr 24;51(16):3485-96
22471521
Annu Rev Biochem. 1993;62:685-713
7688945
Nature. 2007 Dec 13;450(7172):964-72
18075575
Biochemistry. 2004 Nov 16;43(45):14317-24
15533035
Biophys Chem. 1983 Sep;18(2):125-32
6626685
Biochemistry. 1999 Jun 15;38(24):7696-709
10387009
J Biol Chem. 2013 Apr 19;288(16):11590-600
23463511
PLoS Biol. 2009 Oct;7(10):e1000225
19859523
Biochim Biophys Acta. 2011 Aug;1814(8):942-68
21059410
Structure. 2004 Dec;12(12):2125-36
15576027
Biochemistry. 2001 Jun 19;40(24):7228-38
11401570
Biomol NMR Assign. 2010 Oct;4(2):207-9
20582735
Curr Biol. 2004 May 25;14(10):R389-91
15186765
Biochemistry. 2004 Feb 24;43(7):2116-25
14967051
Protein Sci. 2000 Jan;9(1):10-9
10739242
Cell. 2001 Oct 5;107(1):91-102
11595188
Structure. 1999 Feb 15;7(2):R31-5
10368292
J Mol Biol. 1998 Apr 24;278(1):147-65
9571040
Biophys J. 2010 Mar 17;98(6):L15-7
20303846
Biochemistry. 2014 Mar 25;53(11):1768-78
24568554
Proc Natl Acad Sci U S A. 1958 Feb;44(2):98-104
16590179
Biochemistry. 2003 Sep 2;42(34):10200-11
12939148
Biochemistry. 1991 Jan 15;30(2):526-37
1846299
PLoS Comput Biol. 2014 Sep 04;10(9):e1003804
25188490
Biochemistry. 2006 Aug 15;45(32):9675-87
16893169
Adv Protein Chem. 2005;71:401-40
16230118
Mol Cell. 2005 Aug 5;19(3):345-55
16061181
Science. 2003 Aug 29;301(5637):1196-202
12947189
Biochemistry. 2004 Feb 24;43(7):2106-15
14967050
J Biol Chem. 2008 Dec 26;283(52):36711-23
18984592
Chem Rev. 2006 Jun;106(6):2092-110
16771444
J Biol Chem. 2012 Apr 13;287(16):13040-7
22362779
J Biol Chem. 2009 Jul 31;284(31):21090-9
19515847
News Physiol Sci. 2001 Aug;16:171-3
11479367
J Mol Biol. 2008 May 30;379(2):317-30
18448122
Mol Cell. 2004 Mar 12;13(5):751-62
15023344
Nucleic Acids Res. 2007;35(18):6052-62
17766250
Trends Biochem Sci. 2010 Oct;35(10):539-46
20541943
Biochemistry. 2007 Sep 25;46(38):10790-803
17725324