Nucleic Acids Research, Vol 27, Issue 17 3481-3486, Copyright © 1999 by Oxford University Press
LC Kroutil and TA Kunkel
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.
ARTICLES
Deletion errors generated during replication of CAG repeats
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
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