Hypermutation at A/T sites during G.U mismatch repair in vitro by human B-cell lysates.
Somatic hypermutation in the variable regions of immunoglobulin genes is required to produce high affinity antibody molecules. Somatic hypermutation results by processing G.U mismatches generated when activation-induced cytidine deaminase (AID) deaminates C to U. Mutations at C/G sites are targeted mainly at deamination sites, whereas mutations at A/T sites entail error-prone DNA gap repair. We used B-cell lysates to analyze salient features of somatic hypermutation with in vitro mutational assays. Tonsil and hypermutating Ramos B-cells convert C-->U in accord with AID motif specificities, whereas HeLa cells do not. Using tonsil cell lysates to repair a G.U mismatch, A/T and G/C targeted mutations occur about equally, whereas Ramos cell lysates make fewer mutations at A/T sites (approximately 24%) compared with G/C sites (approximately 76%). In contrast, mutations in HeLa cell lysates occur almost exclusively at G/C sites (> 95%). By recapitulating two basic features of B-cell-specific somatic hypermutation, G/C mutations targeted to AID hot spot motifs and elevated A/T mutations dependent on error-prone processing of G.U mispairs, these cell free assays provide a practical method to reconstitute error-prone mismatch repair using purified B-cell proteins.
Pham
Phuong
P
Department of Biological Sciences and Chemistry, University of Southern California, Los Angeles, California 90089-2910, USA.
Zhang
Ke
K
Goodman
Myron F
MF
D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D053843:DNA Mismatch Repair / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics
J Biol Chem. 1996 May 31;271(22):12767-74
8662714
Immunity. 1998 Jul;9(1):105-14
9697840
Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13986-91
16172387
Nat Immunol. 2001 Jun;2(6):530-6
11376340
Nat Immunol. 2001 Jun;2(6):537-41
11376341
Science. 2004 Jul 30;305(5684):645
15286366
Nat Struct Mol Biol. 2004 May;11(5):435-42
15098018
J Exp Med. 2004 Jul 5;200(1):61-8
15238605
J Exp Med. 2004 Jan 19;199(2):265-70
14734526
J Biol Chem. 2006 Oct 13;281(41):30305-9
16905530
DNA Repair (Amst). 2007 Apr 1;6(4):398-409
17129767
J Exp Med. 2005 Apr 18;201(8):1191-6
15824086
Cell. 2003 Jun 13;113(6):803-9
12809610
J Exp Med. 2003 May 19;197(10):1291-6
12756266
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3843-8
18292225
Immunity. 1998 Jul;9(1):135-41
9697843
J Mol Biol. 2004 Mar 26;337(3):585-96
15019779
Nature. 2003 Apr 17;422(6933):726-30
12692563
J Immunol. 1997 Oct 1;159(7):3347-53
9317133
Mol Cell. 2004 Oct 22;16(2):163-71
15494304
J Exp Med. 2003 Oct 20;198(8):1171-8
14568978
Cold Spring Harb Symp Quant Biol. 2000;65:143-55
12760029
Annu Rev Biochem. 2005;74:681-710
15952900
DNA Repair (Amst). 2007 Jun 1;6(6):689-92; author reply 693-4
17291835
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):4102-7
12651944
J Exp Med. 2007 Jan 22;204(1):17-23
17190840
Adv Exp Med Biol. 2007;596:93-109
17338179
Adv Immunol. 2007;94:127-55
17560274
Proc Natl Acad Sci U S A. 2002 May 14;99(10):7003-8
12011459
Immunity. 2001 May;14(5):643-53
11371365
Nat Rev Immunol. 2004 Nov;4(11):868-77
15516966
J Exp Med. 2007 Aug 6;204(8):1989-98
17664295
Mol Immunol. 2007 Apr;44(10):2659-66
17240451
Nat Immunol. 2004 Feb;5(2):224-9
14716311
J Exp Med. 2004 May 3;199(9):1235-44
15117971
Mol Biol Evol. 2005 Feb;22(2):367-77
15496550
Nature. 2005 Nov 24;438(7067):508-11
16251902
Cell. 2000 Sep 1;102(5):553-63
11007474
DNA Repair (Amst). 2008 Aug 2;7(8):1392-8
18562254
Int J Hematol. 2006 Apr;83(3):195-200
16720547
Nature. 2004 Aug 26;430(7003):992-8
15273694
Nat Rev Immunol. 2004 Jul;4(7):541-52
15229473
Nucleic Acids Res. 2002 Feb 1;30(3):E14
11809902
Annu Rev Immunol. 2008;26:481-511
18304001
J Biol Chem. 2001 Nov 9;276(45):42588-600
11551933
EMBO J. 2005 Nov 2;24(21):3757-69
16222339
Mol Cell Biol. 2005 Dec;25(24):10815-21
16314506
Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1975-80
14769937
Nat Struct Mol Biol. 2006 May;13(5):392-9
16622407
Annu Rev Biochem. 2007;76:1-22
17328676
Curr Biol. 2004 Aug 10;14(15):1392-6
15296758
Nucleic Acids Res. 2004 Apr 30;32(8):2421-9
15121899
J Med Dent Sci. 2003 Mar;50(1):41-6
12715918
Curr Biol. 1999 Feb 25;9(4):174-85
10074426
EMBO Rep. 2005 Dec;6(12):1143-8
16319960
Methods Enzymol. 1995;262:232-56
8594351
Eur J Immunol. 1999 Oct;29(10):3122-32
10540323
J Virol. 2001 Nov;75(21):10488-92
11581418
Nucleic Acids Res. 2003 Jun 15;31(12):2990-4
12799424
Nature. 2002 Feb 14;415(6873):802-6
11823785
Immunity. 1996 Jan;4(1):57-65
8574852
J Exp Med. 2000 Feb 7;191(3):579-84
10662804
Proc Natl Acad Sci U S A. 1990 Aug;87(15):5837-41
2116007
J Biol Chem. 1991 Feb 25;266(6):3744-51
1995629
Prog Nucleic Acid Res Mol Biol. 2001;68:165-88
11554295
J Biol Chem. 2007 Jun 15;282(24):17387-94
17449470
Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):9954-9
12119399
Nature. 2003 Jul 3;424(6944):103-7
12819663
J Biol Chem. 2008 Jun 20;283(25):17428-39
18417471
Mol Cell. 2004 May 21;14(4):491-500
15149598
Biochemistry. 2005 Mar 1;44(8):2703-15
15723516
Science. 1987 Nov 20;238(4830):1088-94
3317826
Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8798-803
16723391
J Exp Med. 1998 Jun 1;187(11):1745-51
9607916
J Exp Med. 2006 Feb 20;203(2):319-23
16476771
Annu Rev Immunol. 2008;26:261-92
18370922
J Immunol. 2006 Oct 15;177(8):5386-92
17015724
J Allergy Clin Immunol. 2002 Sep;110(3):421-8
12209089
Cell. 2000 Sep 1;102(5):565-75
11007475
J Biol Chem. 2004 Dec 3;279(49):51612-21
15371439
Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):395-400
16387847
Eur J Immunol. 1993 Jun;23(6):1405-8
7684690
Science. 2007 Aug 31;317(5842):1227-30
17761884
Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8791-4
9671757