Abstract
BackgroundDuring DNA replication or repair, disease-associated (CAG)n/(CTG)n expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Recent studies identified a nick-directed (CAG)n/(CTG)n hairpin repair (HPR) system that removes (CAG)n/(CTG)n hairpins from human cells via endonucleolytic incisions. Because the process is highly similar to the mechanism by which XPG and XPF endonucleases remove bulky DNA lesions during nucleotide excision repair, we assessed the potential role of XPG in conducting (CAG)n/(CTG)n HPR.ResultsTo determine if the XPG endonuclease is involved in (CAG)n/(CTG)n hairpin removal, two XPG-deficient cell lines (GM16024 and AG08802) were examined for their ability to process (CAG)n/(CTG)n hairpins in vitro. We demonstrated that the GM16024 cell line processes all hairpin substrates as efficiently as HeLa cells, and that the AG08802 cell line is partially defective in HPR. Analysis of repair intermediates revealed that nuclear extracts from both XPG-deficient lines remove CAG/CTG hairpins via incisions, but the incision products are distinct from those generated in HeLa extracts. We also show that purified recombinant XPG protein greatly stimulates HPR in XPG-deficient extracts by promoting an incision 5' to the hairpin.ConclusionsOur results strongly suggest that 1) human cells possess multiple pathways to remove (CAG)n/(CTG)n hairpins located in newly synthesized (or nicked) DNA strand; and 2) XPG, although not essential for (CAG)n/(CTG)n hairpin removal, stimulates HPR by facilitating a 5' incision to the hairpin. This study reveals a novel role for XPG in genome-maintenance and implicates XPG in diseases caused by trinucleotide repeat expansion.
Highlights
During DNA replication or repair, disease-associated (CAG)n/(CTG)n expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand
XPG is not essential for (CAG)25 or (CTG)25 hairpin removal Removal of (CTG)n hairpins via dual incision in hairpin repair (HPR) resembles the mechanism by which bulky DNA lesions are cleaved during nucleotide excision repair, where XPG is responsible for the 3’ incision of a lesion
The results show that consistent with the observations in HeLa cells, both XPG mutants removed all containing either a (CAG)/contains a (CAG)25 or (CTG) hairpins via incisions (Figure 5)
Summary
During DNA replication or repair, disease-associated (CAG)n/(CTG)n expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Expansion of trinucleotide repeats (TNRs) is responsible for certain familial neurological, neurodegenerative and neuromuscular disorders, such as CAG repeat expansion-caused Huntington’s disease [1,2,3]. Previous studies have suggested that TNR expansions could result from strand slippage-caused hairpin formations within TNR sequences ( CAG and CTG repeats) in the Recent studies have shown that human cells possess a repair system, referred to as DNA hairpin repair (HPR), that catalyzes error-free removal of CAG/CTG hairpins in a nick-dependent manner [12,13].
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