Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19.
Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5'-GGCGGCGGT/TGGGGCTCG-3' [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.
Jang
Ming Y
MY
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8, Rm. 310, National Institutes of Health, Department of Health and Human Services, 8 Center Dr. MSC 0840, Bethesda, MD 20892-0840, USA.
Yarborough
Orlando H
OH
Conyers
Gary B
GB
McPhie
Peter
P
Owens
Roland A
RA
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*; D016662:Virus Integration
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D004247:DNA
Digestion. 2003;68(1):13-23
12949435
Annu Rev Biochem. 1967;36:407-48
18257727
Eur J Biochem. 1970 Aug;15(2):209-14
5502661
J Virol. 1977 Nov;24(2):627-34
916029
Methods Enzymol. 1980;65(1):499-560
6246368
J Virol. 1983 Feb;45(2):555-64
6300419
Virology. 1989 Jun;170(2):460-7
2543124
Cell. 1990 Jan 12;60(1):105-13
2153052
Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211-5
2156265
Cell. 1990 May 4;61(3):447-57
2159383
J Virol. 1990 Dec;64(12):6204-13
2173787
Genomics. 1991 Jul;10(3):831-4
1653762
EMBO J. 1991 Dec;10(12):3941-50
1657596
J Virol. 1992 Feb;66(2):1119-28
1309894
Biochemistry. 1992 Jan 28;31(3):833-41
1731941
EMBO J. 1992 Dec;11(13):5071-8
1334463
J Virol. 1993 Feb;67(2):997-1005
8380475
Biochemistry. 1993 Jan 19;32(2):598-601
8422371
J Virol. 1993 Oct;67(10):6096-104
8396670
J Virol. 1994 Feb;68(2):797-804
8289383
Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5808-12
8016070
J Virol. 1994 Aug;68(8):4988-97
8035498
J Biol Chem. 1994 Aug 26;269(34):21858-69
8063830
J Virol. 1994 Nov;68(11):7448-57
7933128
Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10039-43
7937833
Nat Genet. 1994 Aug;7(4):472-9
7951316
J Virol. 1995 Apr;69(4):2038-46
7884849
Proc Natl Acad Sci U S A. 1995 May 23;92(11):5199-203
7761473
J Virol. 1995 Nov;69(11):6787-96
7474090
J Virol. 1995 Nov;69(11):6917-24
7474109
J Virol. 1995 Nov;69(11):7334-8
7474165
Gene Ther. 1994 May;1(3):165-9
7584077
J Virol. 1996 Mar;70(3):1542-53
8627673
J Biol Chem. 1996 Mar 1;271(9):5208-14
8617804
J Virol. 1996 Jul;70(7):4783-6
8676507
Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7966-72
8755586
Gene Ther. 1996 Sep;3(9):748-55
8875221
J Virol. 1997 Mar;71(3):2528-34
9032395
J Virol. 1997 Apr;71(4):3299-306
9060699
J Virol. 2000 Mar;74(6):2936-42
10684315
J Virol. 2000 Apr;74(7):3122-9
10708427
J Virol. 2000 May;74(9):3953-66
10756007
J Virol. 2000 Jul;74(13):6213-6
10846109
J Virol. 2000 Aug;74(16):7671-7
10906224
J Virol. 2000 Sep;74(17):7762-71
10933682
J Virol. 2000 Oct;74(19):8831-42
10982325
Arch Biochem Biophys. 2001 May 15;389(2):271-7
11339817
Biochemistry. 2002 Sep 24;41(38):11438-48
12234186
Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12381-5
12221283
Gene Ther. 2003 Sep;10(19):1691-702
12923568
Virology. 2003 Sep 1;313(2):364-76
12954205
J Mol Biol. 1998 Sep 25;282(3):619-36
9737926
J Virol. 1999 Feb;73(2):1309-19
9882336
J Virol. 1999 Feb;73(2):1580-90
9882364
J Virol. 1999 Mar;73(3):2517-26
9971837
J Virol. 1999 Apr;73(4):2682-93
10074114
J Virol. 1999 Apr;73(4):2930-7
10074142
J Virol. 1999 May;73(5):3994-4003
10196295
J Virol. 1999 May;73(5):4293-8
10196327
J Virol. 1999 Oct;73(10):8235-44
10482574
J Virol. 1999 Nov;73(11):9325-36
10516041
Nature. 1953 Apr 25;171(4356):737-8
13054692
Mol Cell. 2004 Feb 13;13(3):403-14
14967147