Mutation versus repair: NEIL1 removal of hydantoin lesions in single-stranded, bulge, bubble, and duplex DNA contexts

Abstract

Human DNA glycosylase NEIL1 exhibits a superior ability to remove oxidized guanine lesions guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) from duplex DNA in comparison to other substrates. In this work, Gh and Sp lesions in bubble, bulge, and single-stranded DNA were found to be good substrates for NEIL1 but were typically excised at much slower rates than from canonical duplex substrates. A notable exception was the activity of NEIL1 on removal of Gh in bubble structures which approaches that of the normal duplex substrate. The cleavage of Gh in the template strand of a replication or transcription bubble may prevent mutations associated with Gh during replication or transcription. However, removal of hydantoin lesions in the absence of an opposite base may also result in strand breaks and potentially deletion and frameshift mutations. Consistent with this as a potential mechanism leading to an N-1 frameshift mutation, the nick left after the removal of the Gh lesion in a DNA bulge by NEIL1 was efficiently religated in the presence of polynucleotide kinase (PNK) and human DNA ligase III (Lig III). These results indicate that NEIL1 does not require a base opposite to identify and remove hydantoin lesions. Depending on the context, the glycosylase activity of NEIL1 may stall replication and prevent mutations or lead to inappropriate removal that may contribute to the mutational spectrum of these unusual lesions.

Figure 1

DNA lesion structures and contexts used in glycosylase assays. A) Chemical structures of lesions examined in various contexts B) A six-nucleotide mispair was used to form a bubble in the middle of a 30-nucleotide duplex. For the bulge-containing duplex, a 29-nucleotide complement was annealed with the lesion-containing 30-nucleotide strand to form a bulge in the middle of the duplex as shown.

Figure 2

Characterization of bubble, bulge, single-stranded and double-stranded oligonucleotides by non-denaturing gel electrophoresis. Lanes 1 to 4 represent [γ-³²P]-ATP labeled oligonucleotides without lesions. Lane 5–8 represent [γ-³²P]-ATP labeled oligonucleotides with the Gh lesion at position X (see Fig. 1). Lanes 1 and 5 are single-stranded DNA. Lanes 2 and 6 are double-stranded DNA. Lane 3 and 7 are bubble structures. Lane 4 and 8 are bulge structures.

Figure 3

Probing structures of bubble and bulge substrates with Mung Bean Nuclease (MBN). Lane 4 shows that double-stranded DNA cannot be cleaved by MBN. Lane 6 shows the mispaired part in the bubble structure can be cleaved by MBN to shorter oligonucleotides (12-nts to 18-nts). Lane 8 shows that in the bulge structure, Gh-containing oligonucleotides can be cleaved by MBN to yield a 14-nt oligonucleotide.

Figure 4

Storage phosphor autoradiograms of NEIL1-mediated excision of hydantoin lesions in single-stranded 30-mer oligonucleotides. A: Gh-containing ss-DNA over a 20-min time course; B: Sp1-containing ss-DNA over a 20-min time course; C, Sp2-containing ss-DNA over a 60-min time course. All reactions were performed at 37°C.

Figure 5

Representative plot of product for by excision of Gh from the bulge structure by NEIL1 over a 60-min period at 37°C. The reaction proceeds to 85% completion in 2 min.

Figure 6

Reconstitution of BER with NEIL1, PNK and Lig III. NEIL1 cleaved the Gh-containing bulge structure and left a single-strand nick with phosphate groups on both 5’and 3’ termini (lanes 2 and 6). With extra MgCl₂ added, PNK removed the phosphate group on the 3’end of the nick, thus preparing the single-nucleotide deletion for ligation (lane 7). Lig III then ligated the two strands to form a 29-mer oligonucleotide (lane 8).