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Deletion errors generated during replication of CAG repeats
1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
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.
Insights
Triplet repeat instability, linked to diseases, involves DNA polymerase deletions. These polymerases create mutations by misaligning triplet repeats, with proofreading offering limited protection.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Triplet repeat sequence instability is a known factor in hereditary neurological disorders and cancers.
- This instability can manifest as deletions of triplet repeats during DNA replication by polymerases.
Purpose of the Study:
- To investigate the mechanism of triplet repeat deletion during DNA replication by specific DNA polymerases.
- To quantify the frequency of deletions versus substitutions at triplet repeat sequences.
- To assess the role of proofreading activity in mitigating triplet repeat instability.
Main Methods:
- Constructed M13mp2 DNA substrates containing (CAG)9 and (CAG)17 repeats with a TAG codon.
- Copied these templates using DNA polymerase beta and exonuclease-deficient T7 DNA polymerase.
- Scored errors as blue plaque Lac revertants and sequenced DNA to identify substitutions or deletions.
Main Results:
- DNA polymerase beta and exonuclease-deficient T7 DNA polymerase generated deletions of 1–8 repeats.
- These polymerases utilized misaligned template-primers, leading to deletions of 3 to 45 nucleotides.
- Deletion frequencies significantly exceeded substitution frequencies, especially without error correction.
- Proofreading-proficient T7 DNA polymerase showed 2- to 10-fold lower deletion frequencies.
Conclusions:
- Triplet repeat sequences are highly susceptible to mutation due to deletions.
- Misaligned triplet repeat sequences are subject to polymerase proofreading, though with reduced efficiency.
- This highlights the risk of triplet repeat expansion/contraction in disease pathogenesis.