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. 2004 Aug 13;32(14):4332-9.
doi: 10.1093/nar/gkh758. Print 2004.

N-terminus of the rat adenine glycosylase MYH affects excision rates and processing of MYH-generated abasic sites

Huaxian Ma  1 , Heung M Lee, Ella W Englander
Affiliations

N-terminus of the rat adenine glycosylase MYH affects excision rates and processing of MYH-generated abasic sites

Huaxian Ma et al. Nucleic Acids Res. .

Abstract

Repair of most modified and mispaired bases in the genome is initiated by DNA glycosylases, which bind to their respective targets and cleave the N-glycosyl bond to initiate base excision repair (BER). The mammalian homolog of the Escherichia coli MutY DNA glycosylase (MYH) cleaves adenine residues paired with either oxidized or non-modified guanines. MYH is crucial for the avoidance of mutations resulting from oxidative DNA damage. Multiple N-terminal splice variants of MYH exist in mammalian cells and it is likely that different variants result in the production of enzymes with altered properties. To investigate whether modifications in the N-terminus are consequential to MYH function, we overexpressed intact and N-terminal-deletion rat MYH proteins and examined their activities. We found that deletion of 75 amino acids, which perturbs the catalytic core that is conserved with E.coli MutY, abolished excision activity. In contrast, deletions limited to the extended mammalian N-terminal domain, differentially influenced steady-state excision rates. Notably, deletion of 50 amino acids resulted in an enzyme with a significantly lower K(m) favoring formation of excision products with 3'-OH termini. Our findings suggest that MYH isoforms divergent in the N-terminus influence excision rates and processing of abasic sites.

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Figures

Figure 1
Figure 1
Sequence alignment of N-terminal amino acids of mammalian MYH and E.coli MutY proteins. Horizontal arrows indicate the N-terminal deletions in the rat sequence (Δ25, Δ50, Δ75). Closed vertical arrowheads indicate the human and mouse MYH variants. Open arrowheads indicate predicted cleavage sites for mitochondrial import in the rat (downwards), human (upwards left) and mouse (upwards right) proteins. Proposed mitochondrial localization signal (MLS) and RPA binding motif are marked. Potential phosphorylation and acetylation sites conserved in all mammalian proteins are highlighted in boldface. A stretch of amino acids corresponding to the α1 helix in MutY (22) is indicated.
Figure 2
Figure 2
Gel migration analysis of 3′ termini generated by MutY, APE and Nfo. Autoradigram shows migration of GO/A excision products generated by MutY alone or in combination with either APE or Nfo and terminated either with or without NaOH. Migration of the product generated by MutY alone represents an abasic site hydrolyzed by NaOH-mediated β-elimination (lane 2, arrow). Addition of APE (20-fold molar excess) in the absence of MgCl2, increased MutY product amount (lane 3), whereas addition of APE in the presence of MgCl2 and termination without NaOH, generated a fast migrating product, consistent with APE cleavage of the phosphodiester bond 5′ to the abasic site and generation of 3′-OH ends (lane 12, arrow). In contrast, addition of Nfo, which is independent of MgCl2, generated the fast migrating product consistent with 3′-OH ends, under all tested conditions (lane 4, 7, 10, 13, arrow). Action of MutY alone generates an abasic site, which is not cleaved in absence of alkali and, accordingly, no product is observed (lane 8 and 11).
Figure 3
Figure 3
Excision products of FL and N-ter deletion-MYH proteins on GO/A and G/A substrates. (A) Expression and subcellular partition of FL and Δ25, Δ50 and Δ75 proteins: western blotting analysis of nuclear (NE), mitochondrial (MT) and cytoplasmic (C) fractions of FL, Δ25, Δ50 and Δ75 with the αFlag antibody and of non-transfected and FL-transfected 293 cells with αVDAC, as indicated. (B) Autoradiogram showing differences in migration and levels of excision products generated on GO/A and G/A substrates incubated with indicated nuclear extracts. Product generated by the E.coli MutY protein is included as size reference (lanes 1 and 7). Low level of excision is seen in naive 293 cells with both substrates (lanes 2 and 8, lower panel, long exposure). Mammalian MYH proteins generate higher levels of products on GO/A than on G/A, while the opposite is observed with E.coli MutY (lanes 1 and 7). On GO/A, the FL protein generates primarily a slowly migrating product (lane 3), Δ25 generates similar amounts of slow and fast products (lane 4), whereas Δ50 generates primarily the fast product (lane 5). Product levels for Δ75 are similar to those observed with naive 293 cells (lane 6). In all cases, only the fast migrating products are observed with the G/A substrate. (C) Bars represent fast (black) and slow (blank) cleavage products generated in three independent assays and expressed as mean ± SEM of radioactivity quantified on the Phosphorimager.
Figure 4
Figure 4
Binding of FL- and N-ter-deletion MYH proteins to GO/A substrate. (A) Autoradiograms show EMSAs with nuclear extracts from naive 293 cells and cells transfected with FL and N-terminal deletion MYH proteins. Complexes formed with MutY are shown for reference (asterisk). Arrowheads indicate specific complexes. Naive 293 cells generated several complexes; only one complex was competed with a 150-fold excess of the GO/A substrate (lanes 25–27). The complex generated with FL was challenged by competition with GO/A, GO/C G/A or G/C (lanes 2–8). A 150-fold molar excess of cold GO/A eliminated the complex formed with Δ50 (lanes 12–13, 19–20) and to a lesser degree complexes formed with Δ25 and FL MYH (lanes 9, 10 and 2, 3, respectively). Cells overexpressing Δ75 show a faint complex (lane 22) similar to that observed with naive 293 cells (lane 25). (B) Binding of immunoprecipitated FL- and N-ter-deletion MYH proteins. Autoradiogram shows complexes formed with eluates of immunoprecipitated FL, Δ25 and Δ50 proteins. Identity of complexes is confirmed by competition with excess specific and non-specific substrates, as indicated (arrowheads). No specific complexes are detectable with Δ75 protein.
Figure 5
Figure 5
Steady-state kinetics of GO/A cleavage by MYH proteins. Representative autoradiograms show excision products generated on GO/A by nuclear extracts from 293 cells transfected with FL, Δ25 or Δ50 MYH. (A) Excision in the presence of MgCl2. Reactions included 0.25 nM end-labeled GO/A* supplemented with increasing concentrations of cold GO/A (0.25–512 nM) as indicated. Product generated by 0.1 U of E.coli MutY served as size reference. Reactions were carried out for 10 min at 37°C. (B) Excision in the absence of MgCl2. Reactions were carried out over the same range of substrate concentrations. E.coli MutY was used at 0.025 U. (C) Steady-state rates of GO/A Cleavage by FL- and N-ter-deletion MYH proteins. Heavy lines show cleavage in the presence of 1 mM MgCl2, regular lines in the absence of MgCl2 and dotted lines in absence of MgCl2 and presence of 2.25 nM EDTA. The values for FL protein are shown as circles, for Δ25 –triangles, and for Δ50 diamonds. Kinetic values represent means of four independent experiments. Standard errors for the data points were in the range of 3–10% (data not shown).
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References

    1. Beckman K.B. and Ames,B.N. (1997) Oxidative decay of DNA. J. Biol. Chem., 272, 19633–19636. - PubMed
    1. Mitra S., Hazra,T.K., Roy,R., Ikeda,S., Biswas,T., Lock,J., Boldogh,I. and Izumi,T. (1997) Complexities of DNA base excision repair in mammalian cells. Mol. Cell, 7, 305–312. - PubMed
    1. Dianov G., Bischoff,C., Piotrowski,J. and Bohr,V.A. (1998) Repair pathways for processing of 8-oxoguanine in DNA by mammalian cell extracts. J. Biol. Chem., 273, 33811–33816. - PubMed
    1. Michaels M.L., Cruz,C., Grollman,A.P. and Miller,J.H. (1992) Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc. Natl Acad. Sci. USA, 89, 7022–7025. - PMC - PubMed
    1. Michaels M.L., Tchou,J., Grollman,A.P. and Miller,J.H. (1992) A repair system for 8-oxo-7,8-dihydrodeoxyguanine. Biochemistry, 31, 10964–10968. - PubMed

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