Deletion errors generated during replication of CAG repeats

Abstract

Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer. Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases. To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes. Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions. DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats. Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides. Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction. Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative. This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches.

Hijacking of the mismatch repair system to cause CAG expansion and cell death in neurodegenerative disease

“…However, as the leading strand template, 5'-CTG or 5' CGG repeats are relatively stable [31,[49][50][51][52][53][54][60][61][62][63][64][65]. Thus, it is not obvious why slippage should be limited to only one orientation if the same sequence is being copied [66]. If some aspect of the lagging strand configuration promotes slippage, there is no compelling explanation for why it should be limited to the template strand.…”

A matter of life or death: How microsatellites emerge in and vanish from the human genome

“…Experimentally, interruptions have been shown to affect microsatellite mutational behavior in a manner consistent with microsatellite death. Interruptions can reduce DNA polymerase slippage-mediated errors in vitro (Kroutil and Kunkel 1999). Moreover, interruptions reduce the rate of microsatellite mutagenesis in vivo from two-to 90-fold, depending on the type and position of the interruption (Petes 1997;Rolfsmeier and Lahue 2000;Boyer 2008).…”

Variant high-molecular-weight glutenin subunits arising from biolistic transformation of wheat

“…Such changes are difficult to assess by MS/MS because these regions are large and devoid of the protease cleavage sites commonly used to fragment proteins for MS. The central repetitive domains are known to be subject to length changes during evolution of native HMW-GS genes, perhaps by homologous recombination between misaligned repeated DNA segments (D'Ovidio et al, 1996;Shewry et al, 1989) or by slippage mistakes made by DNA polymerases during replication (Kroutil and Kunkel, 1999). Some of the same mechanisms may be operative during the introduction of DNA into the wheat embryo cells via biolistics.…”