The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo.
To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Delta324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.
Liu
Minmin
M
Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0086, USA.
Bandaru
Viswanath
V
Bond
Jeffrey P
JP
Jaruga
Pawel
P
Zhao
Xiaobei
X
Christov
Plamen P
PP
Burrows
Cynthia J
CJ
Rizzo
Carmelo J
CJ
Dizdaroglu
Miral
M
Wallace
Susan S
SS
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005720:Gamma Rays; D006146:Guanidines / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006827:Hydantoins / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011743:Pyrimidines / Q000378:metabolism; D012545:Schiff Bases / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013141:Spiro Compounds / Q000378:metabolism; D013379:Substrate Specificity / Q000528:radiation effects; D014633:Valine / Q000378:metabolism
D006146:Guanidines; D006827:Hydantoins; D011743:Pyrimidines; D012545:Schiff Bases; D013141:Spiro Compounds; C467338:guanidinohydantoin; C408822:spiroiminodihydantoin; D006151:Guanosine; C071023:2,6-diamino-4-hydroxy-5-formamidopyrimidine; C039544:4,6-diamino-5-N-formamidopyrimidine; D004247:DNA; D004706:Endodeoxyribonucleases; C540052:NEIL3 protein, mouse; D045647:DNA Glycosylases; D014633:Valine