UVM Theses and Dissertations
Format:
Print
Author:
Liu, Minmin
Dept./Program:
Microbiology and Molecular Genetics
Year:
2012
Degree:
PhD
Abstract:
To protect cells from oxidative DNA damage and mutagenesis, organisms possess mUltiple DNA glycosylases to recognize damaged bases and to initiate the Base Excision Repair (BER) 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 Nei13 protein remain to be elucidated. Previous attempts to express and purify the mouse and human orthologs of Nei13 in theiractive form have not beensuccessful.
In this study, bicistronic expression vectors were constructed for expressing the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3[delta]324), as well as the glycosylase domain of the human NEIL3 (NEIL3[delta]324) in Escherichia coli. It was found that N-terminal initiator methionine processing is critical for the activity of mouse and human proteins. Co-expressing an Escherichia coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNei13, MmuNei13delta]324 and NEIL3[delta]324 improves the N-terminal methionine processing and increases tile percentage of active Nei13 in the preparation. Purified Nei13 proteins are suitable for biochemical, structural and functional studies.
Next, the characterization of a mouse ortholog of Neil3 as an active DNA glycosylase both in vitro and in vivo was reported. 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-formamido pyrimidine (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 an N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine.
We expressed the glycosylase domain of MmuNei13 (MmuNei13[delta]324) 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 Nei13 plays a role in repairing FapyG in vivo. Moreover, the crystal structure of the glycosylase domain of MmuNeil3 (MmuNeil3[delta]324) was determined. Although the structure of MmuNeil3[delta]324 exhibits the same overall fold as other Fpg/Nei proteins, it has two distinct features.
First, MmuNeil3[delta]324 lacks the loop corresponding to the "[alpha]F-[Beta]9/10 loop" of Fpg enzymes, which has been reported to stabilize the flipped-out 8-oxoG in the active site. Secondly, MmuNei13[delta]324 not only lacks two of the three canonical void-filling residues that have been shown to stabilize the opposite strand but it also harbors negatively charged residues on each side of the damaged strand binding cleft, creating a repulsive force for the opposite strand. These distinct structural features of MmuNeil3 provide insight into the substrate specificity of Neil3 as well as its preference for DNA with single-stranded regions.
In this study, bicistronic expression vectors were constructed for expressing the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3[delta]324), as well as the glycosylase domain of the human NEIL3 (NEIL3[delta]324) in Escherichia coli. It was found that N-terminal initiator methionine processing is critical for the activity of mouse and human proteins. Co-expressing an Escherichia coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNei13, MmuNei13delta]324 and NEIL3[delta]324 improves the N-terminal methionine processing and increases tile percentage of active Nei13 in the preparation. Purified Nei13 proteins are suitable for biochemical, structural and functional studies.
Next, the characterization of a mouse ortholog of Neil3 as an active DNA glycosylase both in vitro and in vivo was reported. 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-formamido pyrimidine (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 an N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine.
We expressed the glycosylase domain of MmuNei13 (MmuNei13[delta]324) 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 Nei13 plays a role in repairing FapyG in vivo. Moreover, the crystal structure of the glycosylase domain of MmuNeil3 (MmuNeil3[delta]324) was determined. Although the structure of MmuNeil3[delta]324 exhibits the same overall fold as other Fpg/Nei proteins, it has two distinct features.
First, MmuNeil3[delta]324 lacks the loop corresponding to the "[alpha]F-[Beta]9/10 loop" of Fpg enzymes, which has been reported to stabilize the flipped-out 8-oxoG in the active site. Secondly, MmuNei13[delta]324 not only lacks two of the three canonical void-filling residues that have been shown to stabilize the opposite strand but it also harbors negatively charged residues on each side of the damaged strand binding cleft, creating a repulsive force for the opposite strand. These distinct structural features of MmuNeil3 provide insight into the substrate specificity of Neil3 as well as its preference for DNA with single-stranded regions.