Genetic and biochemical characterization of human AP endonuclease 1 mutants deficient in nucleotide incision repair activity.
BACKGROUND
Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.


RESULTS
We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3'-->5' exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase beta and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.


CONCLUSIONS
Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.
Gelin
Aurore
A
CNRS UMR8126, Université Paris-Sud, Institut de Cancérologie Gustave Roussy, Villejuif, France.
Redrejo-Rodríguez
Modesto
M
Laval
Jacques
J
Fedorova
Olga S
OS
Saparbaev
Murat
M
Ishchenko
Alexander A
AA
D000478:Alkylation; D001483:Base Sequence; D055162:Biocatalysis; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics* / Q000378:metabolism*; D003839:Deoxyadenosines / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*
D003839:Deoxyadenosines; D050505:Mutant Proteins; D009711:Nucleotides; D008741:Methyl Methanesulfonate; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C058118:2'-deoxyadenosine
J Biol Chem. 1998 Nov 13;273(46):30360-5
9804799
DNA Repair (Amst). 2010 Feb 4;9(2):109-19
20006562
Proteins. 2007 Jul 1;68(1):313-23
17427952
DNA Repair (Amst). 2007 Apr 1;6(4):410-28
17208522
Nucleic Acids Res. 2009 Jul;37(12):3946-58
19401441
J Biol Chem. 1998 Nov 13;273(46):30352-9
9804798
N Engl J Med. 2009 Oct 8;361(15):1475-85
19812404
Cell Mol Life Sci. 2008 May;65(10):1544-65
18259689
J Biol Chem. 2003 May 16;278(20):18289-96
12624104
Bioessays. 1999 Aug;21(8):668-76
10440863
DNA Repair (Amst). 2005 Dec 8;4(12):1442-9
16199212
Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2564-9
16473948
Ann N Y Acad Sci. 1994 Jul 29;726:306-8
7522424
Mol Cell. 2005 Feb 4;17(3):463-70
15694346
Nucleic Acids Res. 2004 Jan 02;32(1):73-81
14704345
Biochemistry. 2004 Dec 7;43(48):15210-6
15568813
Curr Opin Struct Biol. 2004 Feb;14(1):43-9
15102448
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13737-41
8943004
Nucleic Acids Res. 2004 May 20;32(9):2844-52
15155853
Mutat Res. 2010 Mar 1;685(1-2):70-9
19751747
Biochemistry. 1999 Apr 20;38(16):4958-64
10213597
Nucleosides Nucleotides Nucleic Acids. 2003 May-Aug;22(5-8):1563-5
14565466
Biochemistry. 1994 Jun 28;33(25):7842-7
7516707
Spectrochim Acta A Mol Biomol Spectrosc. 2001 Jul;57(8):1711-6
11471723
J Biol Chem. 1995 Jul 7;270(27):16002-7
7608159
Nucleic Acids Res. 1995 May 11;23(9):1544-50
7784208
Nature. 2000 Jan 27;403(6768):451-6
10667800
Oncogene. 2002 Dec 16;21(58):8905-25
12483508
EMBO J. 2001 Mar 15;20(6):1477-82
11250913
Biochemistry. 2004 Aug 31;43(34):11017-26
15323560
Mutat Res. 1994 Apr 1;306(1):35-44
7512201
J Mol Biol. 2003 May 30;329(2):311-22
12758078
EMBO J. 1992 Sep;11(9):3323-35
1380454
Proc Natl Acad Sci U S A. 2010 Mar 23;107(12):5528-33
20212167
J Mol Biol. 2001 Apr 6;307(4):1023-34
11286553
Carcinogenesis. 1998 Aug;19(8):1339-43
9744526
Nucleic Acids Res. 1991 Oct 25;19(20):5519-23
1719477
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8919-23
8799128
EMBO J. 1997 Nov 3;16(21):6548-58
9351835
Mol Cell Biol. 2009 Apr;29(7):1834-54
19188445
J Mol Biol. 2000 May 5;298(3):447-59
10772862
Nucleic Acids Res. 2003 Nov 1;31(21):6344-53
14576322
Mol Cell Biol. 2010 Jan;30(2):366-71
19901076
J Biol Chem. 1981 Apr 10;256(7):3405-14
6259165
J Bacteriol. 1986 Dec;168(3):1120-7
2430946
Carcinogenesis. 1998 Mar;19(3):525-7
9525290
J Mol Biol. 1999 Mar 19;287(1):47-57
10074406
J Biol Chem. 2003 Sep 19;278(38):36242-9
12857737
J Biol Chem. 1996 Apr 19;271(16):9573-8
8621631
Mutat Res. 1998 Oct 21;409(1):17-29
9806499
Nature. 2002 Jan 10;415(6868):183-7
11805838
Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11450-4
1722334
Biochemistry. 2010 May 11;49(18):3786-96
20377204
EMBO J. 1996 Dec 2;15(23):6662-70
8978692
Nucleic Acids Res. 1994 Nov 25;22(23):4884-9
7800476
Annu Rev Biochem. 2004;73:39-85
15189136
DNA Repair (Amst). 2007 Jan 4;6(1):8-18
16978929
Nature. 2002 Feb 7;415(6872):655-9
11832948
J Mol Biol. 1999 Jul 16;290(3):667-84
10395822
J Mol Biol. 2008 May 23;379(1):17-27
18439621
Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5739-43
15824325