TY - JOUR
T1 - NEIL2-initiated, APE-independent repair of oxidized bases in DNA
T2 - Evidence for a repair complex in human cells
AU - Das, Aditi
AU - Wiederhold, Lee
AU - Leppard, John B.
AU - Kedar, Padmini
AU - Prasad, Rajendra
AU - Wang, Huxian
AU - Boldogh, Istvan
AU - Karimi-Busheri, Feridoun
AU - Weinfeld, Michael
AU - Tomkinson, Alan E.
AU - Wilson, Samuel H.
AU - Mitra, Sankar
AU - Hazra, Tapas K.
N1 - Funding Information:
This research was supported by USPHS grants CA102271 (TKH), PHS grants CA92584 (SM and AET) and CA81063 (SM), ES012512 (AET), Canadian Institute of Health Research Grant MOP15385 (MW) P01 AG021830 (IB and SM) and P01 ES06676 (TKH, IB and SM). We are grateful to Drs. Miaw-Sheue and P.K. Cooper (Lawrence Berkeley Laboratory) for the production of recombinant NEIL2 baculoviruses. We acknowledge the generous help of Drs T. Wood and A. Kurosky of the NIEHS Center for generating the expression plasmids and MS analysis of the recombinant proteins, respectively. We also thank Dr. David Konkel for editing and Ms. Wanda Smith for secretarial help of this manuscript.
PY - 2006/12/9
Y1 - 2006/12/9
N2 - DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3′ phospho α,β-unsaturated aldehyde (3′ PUA) or 3′ phosphate (3′ P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3′ blocking groups (3′ PUA and 3′ P) to generate the 3′-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3′ PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3′ phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3′ phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase β (Pol β), DNA ligase IIIα (Lig IIIα) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol β and Lig IIIα. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol β, Lig IIIα and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.
AB - DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3′ phospho α,β-unsaturated aldehyde (3′ PUA) or 3′ phosphate (3′ P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3′ blocking groups (3′ PUA and 3′ P) to generate the 3′-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3′ PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3′ phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3′ phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase β (Pol β), DNA ligase IIIα (Lig IIIα) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol β and Lig IIIα. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol β, Lig IIIα and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.
KW - Base excision repair
KW - DNA glycosylase
KW - NEIL2
KW - Oxidative DNA damage
KW - PNK-dependent repair
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U2 - 10.1016/j.dnarep.2006.07.003
DO - 10.1016/j.dnarep.2006.07.003
M3 - Article
C2 - 16982218
AN - SCOPUS:33751102395
SN - 1568-7864
VL - 5
SP - 1439
EP - 1448
JO - DNA Repair
JF - DNA Repair
IS - 12
ER -