Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

Abstract

Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the base excision repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of Escherichia coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity with the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5'2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the "zincless finger" motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNei1. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates.

Fig. 1. Sequence alignment of endonuclease VIII-like proteins (NEIL) with the representative members of Fpg/Nei family

Numbers in parenthesis represent residues not shown between the regions. For MvNei2, the N-terminal 43 aa extension is not shown. The conserved proline and arginine residues important for the DNA glycosylase activity of Fpg/Nei members are denoted by an asterix. Positions where residues are inserted in MvNei1, MvNei2, ZmoNei and NEIL2 are shown by a filled triangle (▲). The DNA binding helix-two turns-helix (H2TH) motif and the β-hairpin loop of the zinc finger motif are boxed in green and orange respectively. The “zincless finger” motif in human NEIL1 is highlighted in blue. The four cysteines that coordinate the zinc atom are shown in cyan except for ZmoNei, MvNei2 and human NEIL2 where the second cysteine is replaced by a histidine colored purple. Dashed redline demarcates “zincless finger” from zinc finger-containing proteins. AtFpg1: Arabidopsis thaliana MMH-1 (gi|18404050); CalNei: Candida albicans Nei (gi|3850130); PtoNei: Psychroflexus torquis Nei (gi|91215880); human NEIL1 (gi|13375817); MvNei1: Mimivirus Nei1 (gi|55819191); Archaeal Fpg (gi|56295548); E. coli Fpg (gi|16131506); E. coli Nei (gi|16128689); ZmoNei: Zymomonas mobilis Nei (gi|56552083); human NEIL2 (gi|21450800); MvNei2: Mimivirus Nei2 (gi|55819586) and human NEIL3 (gi|19684059).

Fig. 2. DNA glycosylase/lyase activities of mimiviral Nei proteins

A and B, Substrate specificity of MvNei1 and MvNei2 for oxidative DNA damages. C, Activity of MvNei1 and 2 on guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) substrates. D, Lyase activity of MvNei1 and 2 on an AP substrate. For panels A, C and D, ³²P-labeled substrates (25 nM) were incubated with 50 nM of EcoNth, EcoNei, human NEIL1, CalNei, EcoFpg, hOGG1 and increasing concentrations of (50 nM, 125 nM and 250 nM) of MvNei1 and MvNei2 in their respective assay buffers as described in “Experimental Procedures”. In Panel B, 25 nM labeled substrate was incubated with 50 nM MvNei1 and varying concentrations (100 nM, 250 nM and 1.25 μM) of MvNei2. The reaction volumes were normalized to radioactive counts before loading onto a denaturing PAGE gel.

Fig. 2. DNA glycosylase/lyase activities of mimiviral Nei proteins

A and B, Substrate specificity of MvNei1 and MvNei2 for oxidative DNA damages. C, Activity of MvNei1 and 2 on guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) substrates. D, Lyase activity of MvNei1 and 2 on an AP substrate. For panels A, C and D, ³²P-labeled substrates (25 nM) were incubated with 50 nM of EcoNth, EcoNei, human NEIL1, CalNei, EcoFpg, hOGG1 and increasing concentrations of (50 nM, 125 nM and 250 nM) of MvNei1 and MvNei2 in their respective assay buffers as described in “Experimental Procedures”. In Panel B, 25 nM labeled substrate was incubated with 50 nM MvNei1 and varying concentrations (100 nM, 250 nM and 1.25 μM) of MvNei2. The reaction volumes were normalized to radioactive counts before loading onto a denaturing PAGE gel.

Fig. 3. dRPase activity of MvNei1 and 2

A 22-mer substrate containing a 5′-dRP moiety was prepared as described under “Experimental Procedures”. Substrate (10 nM) was treated with 100 nM of EcoNei, human NEIL1, MvNei1 and MvNei2 or 1 unit of human DNA polymerase β. Unreacted 22-mer containing 5′-dRP was stabilized by reducing with NaBH₄ and analyzed on a denaturing PAGE gel.

Fig. 4. Specificities of MvNei1 and 2 for oxidized DNA damages in single stranded DNA

Radioactivly labeled single stranded damage-containing oligonucleotides (25 nM) were incubated at 37°C with 25 nM human NEIL1, MvNei1 and MvNei2 in their respective assay buffers as described in “Experimental Procedures”

Fig. 5. Differential activity of MvNei2 on single and double stranded DNA substrates

MvNei2 (50 nM, 125 nM and 250 nM) was incubated with 25 nM of 5′end labeled single stranded spiroiminodihydantoin (Sp) oligonucleotide and 25 nM of double stranded Sp:C substrate at 37°C for 30 min.

Fig. 6. Activities of human NEIL1 and MvNei1 on single stranded oligonucleotides containing oxidative lesions

10 nM human NEIL1 (empty) or MvNei1 (filled) was incubated with 10 nM, 50 nM and 100 nM single stranded substrates at 37°C for 30 min. Error bars represent standard error of the mean from 3 separate experiments.

Fig. 7. Activity of MvNei1 on damage-containing bubble substrates

A, Substrates with 5OHU in single stranded DNA (ss5OHU), duplex DNA (ds5OHU) or in various size bubbles (B5, B11, B19) were prepared as described under “Experimental Procedures” and analyzed on a 10% native PAGE gel to verify the secondary structure in the substrates. B, 10 nM (filled), 50 nM (gray) or 100 nM (empty) single stranded, double stranded or bubble substrates containing the 5OHU damage were incubated with 10 nM enzyme as described under “Experimental Procedures”. Error bars represent standard error of the mean from 3 separate experiments.

Fig. 7. Activity of MvNei1 on damage-containing bubble substrates

A, Substrates with 5OHU in single stranded DNA (ss5OHU), duplex DNA (ds5OHU) or in various size bubbles (B5, B11, B19) were prepared as described under “Experimental Procedures” and analyzed on a 10% native PAGE gel to verify the secondary structure in the substrates. B, 10 nM (filled), 50 nM (gray) or 100 nM (empty) single stranded, double stranded or bubble substrates containing the 5OHU damage were incubated with 10 nM enzyme as described under “Experimental Procedures”. Error bars represent standard error of the mean from 3 separate experiments.

Fig. 8. Substrate specificity of MvNei1 under steady state conditions

10 nM (black), 50 nM (gray) and 100 nM (empty) of radiolabeled double stranded substrates containing various base damages were incubated with 10 nM MvNei1 at 37°C for 30 min. Error bars represent standard error of the mean from 3 separate experiments.

Fig. 9. Opposite base specificity of MvNei1

10 nM MvNei1 was incubated with 50 nM substrate with A (◇), T (■), G(△), C (●) bases opposite A, thymine glycol (Tg) and B, guanidinohydantoin (Gh) damages at 37°C for various times. Error bars represent standard error of the mean from 3 separate experiments.

Fig. 9. Opposite base specificity of MvNei1

10 nM MvNei1 was incubated with 50 nM substrate with A (◇), T (■), G(△), C (●) bases opposite A, thymine glycol (Tg) and B, guanidinohydantoin (Gh) damages at 37°C for various times. Error bars represent standard error of the mean from 3 separate experiments.

Fig. 10. DNA glycosylase/lyase activities of wild type and mutant MvNei1

A, Radiolabeled Tg:A and AP:G substrates (25 nM) were incubated with 25 nM, 125 nM and 250 nM wild type and mutant MvNei1 enzymes as described under “Experimental Procedures”. B, Effect of R277A mutation on the DNA glycosylase/lyase activity on a Tg:A substrate. 10 nM radiolabeled substrate was incubated with different concentrations of wild type (●) and R277A mutant (▲) as described under “Experimental Procedures”. Error bars represent standard error of the mean from 3 separate experiments.