Abstract
The oxidized DNA base 8-oxoguanine (8-oxoG) is implicated in neuronal CAG repeat expansion associated with Huntington disease, yet it is unclear how such a DNA base lesion and its repair might cause the expansion. Here, we discovered size-limited expansion of CAG repeats during repair of 8-oxoG in a wild-type mouse cell extract. This expansion was deficient in extracts from cells lacking pol beta and HMGB1. We demonstrate that expansion is mediated through pol beta multinucleotide gap-filling DNA synthesis during long-patch base excision repair. Unexpectedly, FEN1 promotes expansion by facilitating ligation of hairpins formed by strand slippage. This alternate role of FEN1 and the polymerase beta (pol beta) multinucleotide gap-filling synthesis is the result of uncoupling of the usual coordination between pol beta and FEN1. HMGB1 probably promotes expansion by stimulating APE1 and FEN1 in forming single strand breaks and ligatable nicks, respectively. This is the first report illustrating that disruption of pol beta and FEN1 coordination during long-patch BER results in CAG repeat expansion.
Highlights
Trinucleotide repeat (TNR)2 expansion has been identified as a cause of more than 40 human neurodegenerative diseases, including Huntington disease (CAG/CTG), myotonic dystrophy type 1 (CTG/CAG), Friedreich ataxia (GAA/TTC), Fragile X syndrome (CGG/CCG), and many others (1, 2)
We went on to explore mechanisms for repeat expansion that involved pol  gap-filling and FEN1 cleavage. It appeared that single-stranded DNA (ssDNA) breaks within CAG repeats resulted in pol  multinucleotide gap-filling DNA synthesis and FEN1 “alternate cleavage,” which promoted CAG repeat expansion
Our results illustrate how the disruption of coordination between pol  and FEN1 during long-patch BER (LP-BER) may lead to CAG repeat expansion and emphasize the critical role of FEN1 processing of the preligation hairpin intermediate
Summary
Materials—DNA oligonucleotides containing 8-oxoG were from Operon Biotechnologies Inc. (Huntsville, AL). In Vitro BER Reconstitution with Purified Enzymes—BER of ssDNA breaks was reconstituted with purified BER enzymes, OGG1, APE1, pol , FEN1, LIG I, or T4 DNA ligase (NEB, Ipswich, MA), and CAG repeat-containing substrates or random DNA sequence substrate with 8-oxoG in one strand. These substrates were preincubated with 1 M OGG1 and 50 nM APE1 at 37 °C for 30 min to create substrates for measuring pol  DNA synthesis activity. APE1 incision of DNA with THF, an abasic site analog, was measured in a 20-l reaction mixture containing 50
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