Substitution of Ala for Tyr567 in RB69 DNA polymerase allows dAMP and dGMP to be inserted opposite Guanidinohydantoin .
Continuous oxidative damage inflicted on DNA produces 7,8-dihydro-8-oxoguanine (8-oxoG), a commonly occurring lesion that can potentially cause cancer by producing G → T transversions during DNA replication. Mild oxidation of 8-oxoG leads to the formation of hydantoins, specifically guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), which are 100% mutagenic because they encode almost exclusively the insertion of dAMP and dGMP (encoding G → T and G → C transversions, respectively). The wild-type (wt) pol α family DNA polymerase from bacteriophage RB69 (RB69pol) inserts dAMP and dGMP with low efficiency when situated opposite Gh. In contrast, the RB69pol Y567A mutant inserts both of these dNMPs opposite Gh with >100-fold higher efficiency than wt. We now report the crystal structure of the "closed" preinsertion complex for the Y567A mutant with dATP opposite a templating Gh (R-configuration) in a 13/18mer primer-template (P/T) at 2.0 Å resolution. The structure data reveal that the Y to A substitution provides the nascent base pair binding pocket (NBP) with the flexibility to accommodate Gh by allowing G568 to move in the major-to-minor groove direction of the P/T. Thus, Gh is rejected as a templating base by wt RB69pol because G568 is inflexible, preventing Gh from pairing with the incoming dATP or dGTP base.
Beckman
Jeff
J
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8024, USA.
Wang
Mina
M
Blaha
Gregor
G
Wang
Jimin
J
Konigsberg
William H
WH
D019943:Amino Acid Substitution; D001435:Bacteriophages / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D006146:Guanidines / Q000378:metabolism*; D006827:Hydantoins / Q000378:metabolism*; D008958:Models, Molecular; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D006146:Guanidines; D006827:Hydantoins; D014764:Viral Proteins; C467338:guanidinohydantoin; C012873:2'-deoxy-5'-adenosine monophosphate; C007257:2'-deoxyguanosine 5'-phosphate; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase
Nucleic Acids Res. 2009 Apr;37(5):1638-49
19153138
Proc Natl Acad Sci U S A. 1986 Aug;83(15):5434-8
3461441
Cancer Res. 1991 Oct 1;51(19):5430-2
1655250
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
Methods Enzymol. 1997;276:307-26
27754618
Org Lett. 2006 Nov 9;8(23):5345-7
17078714
Biochemistry. 2010 Mar 2;49(8):1658-66
20099873
Chem Commun (Camb). 2009 Aug 21;(31):4619-31
19641798
Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6866-70
1495976
Cell. 2001 Jun 1;105(5):657-67
11389835
Cancer Res. 1995 Sep 15;55(18):3982-7
7664267
FASEB J. 1989 Jan;3(1):14-21
2642867
Biochemistry. 2009 Aug 11;48(31):7547-55
19580325
Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):814-8
17210917
Biochemistry. 2000 Oct 24;39(42):12979-88
11041863
Biochemistry. 2007 Aug 21;46(33):9355-65
17655276
Nat Biotechnol. 2004 Jun;22(6):755-9
15156154
J Am Chem Soc. 2007 May 2;129(17):5551-7
17411040
J Am Chem Soc. 2004 Nov 10;126(44):14419-27
15521761
Biochemistry. 2003 Nov 11;42(44):13008-18
14596616
Biochemistry. 2008 Jul 8;47(27):7137-46
18543945
J Phys Chem B. 2008 Dec 25;112(51):16860-73
19049279
Biochemistry. 1987 Dec 15;26(25):8410-7
3327522
J Am Chem Soc. 2004 Feb 4;126(4):1102-9
14746479
DNA Repair (Amst). 2009 May 1;8(5):643-53
19217358
Mutat Res. 2005 Apr 1;571(1-2):33-42
15748636
Mutat Res. 2000 Oct 16;461(2):145-56
11018587
Biochemistry. 2006 Feb 21;45(7):2211-20
16475809
EMBO J. 2004 Apr 7;23(7):1494-505
15057282
J Radiat Res. 2001 Dec;42(4):409-13
11951664
J Biol Chem. 1992 Jul 5;267(19):13320-6
1618833
Nature. 2007 Jun 21;447(7147):941-50
17581577
Environ Health Perspect. 2002 Oct;110 Suppl 5:713-7
12426118
Angew Chem Int Ed Engl. 2008;47(11):2059-62
18260080
Mol Gen Genet. 1994 Jun 3;243(5):500-5
8208241
Nucleic Acids Res. 2001 Oct 15;29(20):4206-14
11600709
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Carcinogenesis. 1991 May;12(5):865-71
2029751
Biochemistry. 2007 Jan 16;46(2):448-60
17209555
Biochemistry. 2010 May 18;49(19):4116-25
20411947
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1194-9
11830658
Biochemistry. 2000 Jun 27;39(25):7613-20
10858312
Biochemistry. 2009 Jul 28;48(29):6940-50
19522539
Biochemistry. 2010 Mar 23;49(11):2502-9
20166752
Chem Res Toxicol. 2001 Jul;14(7):927-38
11453741
Oncogene. 2002 Dec 16;21(58):8895-904
12483507
Biochemistry. 1996 Apr 2;35(13):4026-33
8672436
Biochemistry. 2005 Mar 8;44(9):3338-46
15736944
Nucleic Acids Res. 1999 Jan 15;27(2):496-502
9862971
Biochemistry. 2002 Dec 24;41(51):15304-14
12484769
Mutat Res. 1999 Sep 15;445(1):93-8
10521694
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6597-602
12011423
DNA Repair (Amst). 2005 Jan 2;4(1):41-50
15533836
J Phys Chem B. 2006 Jun 1;110(21):10557-66
16722767
Nucleic Acids Res. 2005 May 06;33(8):2620-8
15879351