Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA.
Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.
Shokri
Leila
L
Department of Physics, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA.
Marintcheva
Boriana
B
Eldib
Mootaz
M
Hanke
Andreas
A
Rouzina
Ioulia
I
Williams
Mark C
MC
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000737:chemistry; D013816:Thermodynamics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; C060035:gp32 protein, Enterobacteria phage T4; D012965:Sodium Chloride; D004247:DNA
J Biol Chem. 2006 Dec 15;281(50):38689-96
17050544
Acc Chem Res. 2002 Mar;35(3):159-66
11900519
Nat Struct Biol. 2000 Aug;7(8):648-52
10932248
Biochemistry. 1980 Jul 22;19(15):3522-30
7407056
Biophys J. 2005 Sep;89(3):1941-56
15994897
J Biol Chem. 1997 Mar 28;272(13):8380-7
9079662
J Mol Biol. 1981 Jan 5;145(1):105-21
7265197
Proteins. 2000;Suppl 4:8-22
11013397
J Biol Chem. 1988 Jul 15;263(20):9831-9
2838482
Biophys J. 2005 Aug;89(2):895-902
15908574
J Mol Biol. 1998 May 22;278(5):999-1014
9600857
Biochemistry. 1977 Nov 1;16(22):4783-90
911789
J Mol Biol. 2005 Jun 3;349(2):317-30
15890198
J Mol Biol. 1974 Jun 25;86(2):469-89
4416620
J Mol Biol. 2004 Feb 27;336(4):851-70
15095865
Annu Rev Biochem. 1994;63:527-70
7979247
J Bacteriol. 2001 Mar;183(6):1862-9
11222583
Biophys J. 2001 Feb;80(2):874-81
11159454
J Biol Chem. 1992 Jul 25;267(21):15022-31
1634538
J Biol Chem. 1998 Mar 13;273(11):6556-64
9497392
Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):1855-60
18238893
Langmuir. 2006 May 9;22(10):4699-709
16649785
Protein Sci. 2004 Jul;13(7):1942-7
15169953
J Biol Chem. 2002 Dec 27;277(52):50643-53
12379653
J Mol Biol. 1981 Oct 15;152(1):67-109
6279865
Mol Gen Genet. 1973 Jul 2;123(3):247-62
4581369
Biochemistry. 2002 May 14;41(19):6032-44
11993998
J Biol Chem. 1985 Mar 25;260(6):3594-603
3882711
J Mol Biol. 2003 Mar 28;327(3):571-8
12634053
Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10173-7
8234273
J Mol Biol. 1981 Jan 5;145(1):75-104
7265204
Curr Opin Struct Biol. 2002 Jun;12(3):330-6
12127451
Biochemistry. 2002 Oct 1;41(39):11611-27
12269804
J Biol Chem. 2003 Aug 8;278(32):29538-45
12766155
Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9557-62
11481454
Biochemistry. 1981 Nov 24;20(24):6929-48
7317363
J Biomol Struct Dyn. 1986 Oct;4(2):251-62
3271443
Biopolymers. 1971;10(12):2623-4
5126533
EMBO J. 1996 Apr 15;15(8):2010-9
8617248
Biochemistry. 2006 Apr 25;45(16):5190-205
16618108
Biopolymers. 1984 Jan;23(1):39-58
6696976
Biophys J. 2001 Feb;80(2):894-900
11159456
Biophys J. 1980 Oct;32(1):403-18
6264988
J Biol Chem. 2004 May 21;279(21):22190-7
15044449
Phys Rev E Stat Nonlin Soft Matter Phys. 2005 Oct;72(4 Pt 1):041102
16383357
Biochemistry. 1981 Nov 24;20(24):6961-77
7032584
J Mol Biol. 2006 May 26;359(1):55-65
16677671
Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2987-92
9096333
J Biol Chem. 1992 Jul 25;267(21):15032-40
1634539
Biochemistry. 1996 Apr 23;35(16):5272-9
8611514
Biochemistry. 1999 Jun 1;38(22):7388-97
10353851
Annu Rev Biochem. 1986;55:103-36
3527040
Mol Microbiol. 2002 Mar;43(6):1505-15
11971263
J Biol Chem. 1994 Feb 18;269(7):5270-8
8106511
Mol Gen Genet. 1981;183(1):66-73
6948996
J Mol Biol. 1981 Jan 5;145(1):123-38
6455528
Proc Natl Acad Sci U S A. 1973 Jun;70(6):1846-50
4578445