Broad segmental progeroid changes in short-lived Ercc1 ^−/Δ7 mice

Abstract

Genome maintenance is considered a prime longevity assurance mechanism as apparent from many progeroid human syndromes that are caused by genome maintenance defects. The ERCC1 protein is involved in three genome maintenance systems: nucleotide excision repair, interstrand cross-link repair, and homologous recombination. Here we describe in-life and post-mortem observations for a hypomorphic Ercc1 variant, Ercc1−/Δ7, which is hemizygous for a single truncated Ercc1 allele, encoding a protein lacking the last seven amino acids. Ercc1−/Δ7 mice were much smaller and median life span was markedly reduced compared to wild-type siblings: 20 and 118 weeks, respectively. Multiple signs and symptoms of aging were found to occur at an accelerated rate in the Ercc1−/Δ7 mice as compared to wild-type controls, including a decline in weight of both whole body and various organs, numerous histopathological lesions, and immune parameters. Together they define a segmental progeroid phenotype of the Ercc1−/Δ7 mouse model.

Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair

“…To verify the presence of an 8-oxoG and abasic site (THF residue) in the loop region of the substrates, we initially probed the formation of hairpins in the CAG repeat tract of the substrates using ssDNA-specific nucleases, Mung Bean Nuclease and S1 Nuclease. We found that Mung Bean Nuclease cleavage on the (CAG) 7 14 -THF hairpin substrate, the nuclease cleavage led to production of products with 45, 48 and 52 nt (Figure 2.3, lanes 7-9), whereas S1 Nuclease cleavage on the (CAG) 14 -8-oxoG and (CAG) 14 -THF hairpin substrates resulted in products with 45, 49, 51 and 54 nt (Figure 2.2 and 2.4, lanes 7-9). This indicates that the substrates contained a hairpin with a (CAG) 4 loop with an 8-oxoG or THF residue and 10 CAG repeats in the stem region.…”

“…Substrates containing a (CAG) 7 or (CAG) 14 hairpin with an 8-oxoG or a tetrahydrofuran (THF), an abasic site analog (used to represent an oxidized sugar in this study), located in the hairpin loop region were designed to mimic a TNR hairpin with a base lesion in the hairpin loop region. Substrates were constructed by annealing the damaged strands that contained (CAG) 13 or (CAG) 20 repeats to their template strand containing (CTG) 7 repeats at a molar ratio of 1:1.…”

“…In vitro BER of an 8-oxoG or oxidized abasic site (THF) in the loop region of CAG repeat hairpins with varying sizes was performed by incubating 25 nM substrates containing a (CAG) 7 or (CAG) 14 Substrates and products were separated by 15% urea-denaturing polyacrylamide gel electrophoresis and detected by a Pharos FX Plus PhosphorImager from Bio-Rad.…”

“…The enzyme removes an 8-oxoG in a CAG hairpin loop that is detached from duplex DNA 700-fold slower than it does in duplex DNA (204). To determine if OGG1 can remove an 8-oxoG in a CAG hairpin loop that is located in duplex DNA, we examined OGG1 cleavage of an 8-oxoG in the loop region of a (CAG) 7 or (CAG) 14 hairpin in a CAG repeat tract. To verify the presence of an 8-oxoG and abasic site (THF residue) in the loop region of the substrates, we initially probed the formation of hairpins in the CAG repeat tract of the substrates using ssDNA-specific nucleases, Mung Bean Nuclease and S1 Nuclease.…”

“…To determine if removal of an 8-oxoG and an abasic lesion located at a CAG repeat hairpin may result in any repaired product, we reconstituted BER for removing a base lesion in the loop region of a (CAG) 7 or (CAG) 14 hairpin substrate. Surprisingly, we found that BER in the small (CAG) 7 hairpin loop generated a product with the same length as the template strand.…”