The in vivo
requirement of OGG1 for expansion of CAG repeats implicated a BER mechanism. Therefore, we tested whether expansion could be regenerated in vitro
using purified human OGG1 and the BER machinery. DNA polymerases can generate expansion on CAG-containing templates by primer extension29–32
and on substrates that mimic BER intermediates32,33
. However, no experiments have tested the entire BER process beginning with the glycosylase incision of a lesion within CAG duplex DNA. We synthesized two DNA templates, each 100 base pairs in length, in which a single 8-oxoG base was positioned 23 nucleotides from the 5′ end of one strand (Supplementary Fig. 6a, b, top panels). In the random template control (Supplementary Fig. 6a), the base lesion was flanked by random sequence DNA of roughly equal CG/AT content, whereas in the CAG template, the sequence was identical except that the base lesion was flanked on the 3′ side by 19 CAG repeats (Supplementary Fig. 6b).
We evaluated the BER pathway using a step-by-step addition of OGG1, apurinic/apirimidinic endonuclease (APE1) and polymerase β (Polβ) to the reconstituted in vitro
system. Removal of the damaged base by OGG1/APE cleavage generates a fragment of 22 nucleotides (Supplementary Fig. 6a, b). Polβ was chosen as the gap-filling polymerase because it is the major BER polymerase in mammalian cells, and because it favours single nucleotide additions34
. If the CAG sequences did not promote expansion, the CAG templates should behave as a random template. Polβ would restore the initial 23 nucleotide length and the template to 100 nucleotides after ligation (Supplementary Fig. 6a). On the other hand, if the CAG sequences promoted expansion, then some strand displacement/slippage should occur. In this case, Polβ should yield multi-nucleotide additions (n
> 1 nucleotide) and templates longer than 100 nucleotides following ligation of looped intermediates (Supplementary Fig. 6b).
With both templates, OGG1 activity was strong, and no difference was observed in the products between the CAG and random templates (, lanes 1 and 4). Addition of APE1 resulted in nicking of the phosphodiester backbone and the production of the 22 nucleotide fragment (, lanes 2 and 5). In the absence of APE, Polβ was unable to carry out strand extension of the 22 nucleotide fragment, as expected (data not shown).
OGG1 excision of 8-oxo-G within CAG repeat DNA can initiate strand displacement and expansion in vitro during BER
We found that gap filling by Polβ on the random template resulted in a single nucleotide addition as the major product, and restored the base at the position 23 nucleotides from the end (, lane 3). In contrast, on CAG templates, OGG1/APE cleavage and gap filling synthesis by Polβ generated longer addition products (, lane 6; black dots). Three nucleotide additions were favoured (, black dots). On random templates, the addition of ligase primarily restored the 100 nucleotide full-length starting material (, lane 2), whereas on the CAG template, ligation resulted in appearance of fragments corresponding to the starting material as well as expanded products (shown in starred bracket ). Thus, OGG1-mediated BER was able to initiate expansion through strand displacement/slippage during the gap-filling step of BER, and both displacement and expansion depended on the CAG sequence.