Kinetic mechanism for DNA unwinding by multiple molecules of Dda helicase aligned on DNA.
Helicases catalyze the separation of double-stranded nucleic acids to form single-stranded intermediates. Using transient state kinetic methods, we have determined the kinetic properties of DNA unwinding under conditions that favor a monomeric form of the Dda helicase as well as conditions that allow multiple molecules to function on the same substrate. Multiple helicase molecules can align like a train on the DNA track. The number of base pairs unwound in a single binding event for Dda is increased from approximately 19 bp for the monomeric form to approximately 64 bp when as many as four Dda molecules are aligned on the same substrate, while the kinetic step size (3.2 +/- 0.7 bp) and unwinding rate (242 +/- 25 bp/s) appear to be independent of the number of Dda molecules present on a given substrate. The data support a model in which the helicase molecules bound to the same substrate move along the DNA track independently during DNA unwinding. The observed increase in processivity arises from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. These results are in contrast to previous reports in which multiple Dda molecules on the same track greatly enhanced the rate and amplitude for displacement of protein blocks on the track. Therefore, only when the progress of the lead molecule in the train is impeded by some type of block, such as a protein bound to DNA, do the trailing molecules interact with the lead molecule to overcome the block. The fact that trailing helicase molecules have little impact on the lead molecule in the train during routine DNA unwinding suggests that the trailing molecules are moving at rates similar to that of the lead molecule. This result implicates a step in the translocation mechanism as contributing greatly to the overall rate-limiting step for unwinding of duplex DNA.
Eoff
Robert L
RL
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Raney
Kevin D
KD
D020029:Base Pairing; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004247:DNA; D004265:DNA Helicases
Nucleic Acids Res. 2008 Apr;36(6):1976-89
18276648
Science. 2007 Jul 27;317(5837):513-6
17656723
Biochemistry. 2005 Jan 18;44(2):666-74
15641792
J Biol Chem. 2004 Jun 18;279(25):26005-12
15087464
Nature. 2004 Jul 22;430(6998):476-80
15269774
Nature. 1987 Mar 19-25;326(6110):312-4
2950327
Nature. 2003 Jun 19;423(6942):889-93
12815437
J Mol Biol. 2004 Oct 8;343(1):83-99
15381422
Nat Rev Mol Cell Biol. 2008 May;9(5):391-401
18414490
J Biol Chem. 2006 Nov 24;281(47):36110-6
16984922
Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3869-73
7731998
EMBO J. 2008 Jun 18;27(12):1718-26
18497749
J Biol Chem. 1994 Dec 16;269(50):31350-8
7989299
J Mol Biol. 2004 Dec 10;344(5):1287-309
15561144
Nat Struct Mol Biol. 2004 Jun;11(6):494-6
15164003
Biochemistry. 2009 Jul 14;48(27):6326-34
19473041
EMBO J. 2008 May 7;27(9):1388-98
18388857
J Mol Biol. 2004 Jun 11;339(4):751-71
15165848
Methods Enzymol. 2009;467:601-626
19897109
EMBO J. 2006 May 17;25(10):2230-9
16642041
Q Rev Biophys. 2003 Feb;36(1):1-69
12643042
Biochemistry. 2005 Oct 4;44(39):12990-7
16185067
J Biol Chem. 2001 Jun 8;276(23):19691-8
11278788
Biochemistry. 1999 Apr 20;38(16):5164-71
10213622
Science. 1997 Jan 17;275(5298):377-80
8994032
Annu Rev Biophys. 2008;37:317-36
18573084
Cell. 1999 Apr 2;97(1):75-84
10199404
J Biol Chem. 1995 Sep 22;270(38):22236-42
7673202
J Mol Biol. 2003 Jan 31;325(5):889-912
12527298
Nucleic Acids Res. 2006 May 31;34(10):3020-9
16738140
Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14722-7
12411580
J Mol Biol. 2002 Nov 29;324(3):409-28
12445778
J Mol Biol. 2001 Jul 6;310(2):327-50
11428893
Biophys J. 2003 Oct;85(4):2224-39
14507688
J Biol Chem. 2009 Jan 23;284(4):2512-21
19010782
J Mol Biol. 2008 Apr 11;377(5):1304-13
18314134
J Biol Chem. 2004 Apr 30;279(18):19035-45
14871889
Eur Phys J E Soft Matter. 2005 May;17(1):7-19
15864723
Nature. 2003 Jun 19;423(6942):893-7
12815438
Cell. 1986 Dec 5;47(5):793-806
3022939
Annu Rev Biochem. 2000;69:651-97
10966472
Nat Struct Mol Biol. 2004 Jun;11(6):531-8
15146172
J Biol Chem. 1995 Mar 31;270(13):7462-73
7706292
Nature. 2006 Jan 5;439(7072):105-8
16397502
J Biol Chem. 2006 Jul 7;281(27):18265-8
16670085
Nature. 2006 Jul 20;442(7100):270-5
16855583
Annu Rev Biochem. 2007;76:23-50
17506634
J Mol Biol. 2003 Jan 31;325(5):913-35
12527299
J Biol Chem. 2007 Sep 14;282(37):27076-27085
17631491
Mol Cell. 2009 Jul 10;35(1):105-15
19595720
Nature. 2004 Nov 11;432(7014):187-93
15538360
Mol Cell. 2007 May 11;26(3):335-47
17499041
Nat Struct Mol Biol. 2006 Mar;13(3):242-9
16474403
Cell. 2006 Dec 29;127(7):1349-60
17190599