br Reaction mechanism The dissimilarity between
Reaction mechanism The dissimilarity between monofunctional and bifunctional glycosylases is that the monofunctional glycosylases removes the substrate base, leaving an intact AP site while the bifunctional glycosylases have an additional lyase activity. This intrinsic lyase activity is present in DNA glycosylases specific for oxidized bases like NEIL1, 2, 3, NTH1 and OGG1. Monofunctional glycosylases normally use an activated water molecule as a nucleophile in attacking sugar C1′ of the damaged nucleotide while bifunctional glycosylases mostly use ε-NH2 of a lysine or the N-terminal proline as the active site nucleophile. The common mechanism of specific nor-Binaltorphimine dihydrochloride recognition by DNA glycosylase is the extrahelical flipping of damaged base into the damaged base-recognition site pocket of enzyme. All DNA glycosylases known so far bind to the minor groove, curve DNA at the site of damage, and flip the lesion base out of the DNA major groove. Thus, the DNA glycosylases are substrate specific, and only those bases that can be accommodated in the specific binding pocket after nucleotide flipping and attain necessary orientation are excised. But the initial recognition is mainly by the structural deformity in DNA caused by the damaged base (Hegde et al., 2008). NEIL1 prefers oxidized pyrimidines created by ROS-derived lesions (Bandaru et al., 2002, Hazraa et al., 2007, Wallace et al., 2003). It shows strong affinity for Fapy-A, Fapy-G and Tg and form a Schiff base with these substrates. The reaction initiates with the recognition of the oxidized/damaged bases. The enzyme then arranges itself in such a way that the damaged base interacts with the catalytic site of the enzyme. The reaction involves a nucleophilic attack at C1′ by a nucleophile in active site which removes the damaged deoxyribose residue to produce a 3′PO4− terminus at the DNA strand break. The nucleophile which catalyzes this reaction is N-terminal proline (Hazraa et al., 2007). It has been found that the mutant protein with mutations in catalytic residues P2T and E3Q lacks the glycosylase activity (Bandaru et al., 2002). Removal of damaged bases causes the formation of an abasic Schiff base intermediate. The intermediate forms stable ‘covalent complex’ by reduction with sodium borohydride (Hazraa et al., 2007). The stability of this covalent complex is essential for the subsequent DNA lyase reaction. Lyase activity may be a single lytic reaction at the 3′ phosphodiester bond (β-elimination) or a consecutive double lytic reaction takes place at both the 3′ and 5′ phosphodiester bond (consecutive β and δ-elimination). The β-lyase reaction produces a single base gap containing 5′-PO4− and 3′-phopho α, β unsaturated aldehyde while the βδ-lyase produces a gap with 3′ and 5′ phosphates (Hazraa et al., 2007). In case of NEIL1, the intermediate undergoes double lyase activity i.e. βδ-elimination reaction and the damaged base is excised producing a SSB that possess phosphates at both 3′ and 5′ ends (Fig. 4) (Hazra et al., 2002a, Hazra et al., 2002b, Mitra et al., 2002). Activity and substrate specificity of NEIL1 largely depends on the DNA structure, and it has significant 5-hydroxyuracil excision activity with single-stranded or bubble DNA (Hegde et al., 2008).
Polymorphisms NEIL1 gene is located on chromosome 15q24.2 spanning 8179bp and contains 11 exons that transcribe in 1828bp mRNA. Exon 1 encodes 1–145, exon 2: 145–185 , exon 3: 185–207, exon 4: 207–240, exon 5: 240–283 , exon 6: 283–292, exon 7: 292–313, exon 8: 313–368 and exon 9: 368–390 amino acids (GeneCards, n.d.). A huge number of SNPs of this gene have been reported in dbSNP database. A total of 996 SNPs are present in different regions of this gene (Fig. 5). Most of the SNPs are present in intronic region (436) while few are present in 3′ (15) and 5′ UTR (162) region. The missense SNPs are 234 in total and constitute 20% of total SNP while synonymous coding SNPs are 124. SNPs in each region of gene have different effects on activity of the protein. SNPs in intronic region may affect the splicing of mRNA. SNPs in 3′ UTR and 5′ UTR regions can alter the binding of miRNAs. Impairment in the structure may induce a reduction in the activity and a reduction in this activity may affect the whole BER process.