Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair

Background:

• DNA sequence and mismatch repair mechanisms are crucial for maintaining genomic integrity.
• Mononucleotide runs are known hotspots for mutations.
• The human MutS homolog 6 (hMSH6) protein plays a key role in mismatch repair.

Purpose of the Study:

• To investigate the role of a specific DNA sequence, a mononucleotide poly(G) run, in mutagenesis.
• To determine the influence of mismatch repair deficiency, specifically in hMSH6, on mutation types.
• To assess the interplay between DNA sequence, mismatch repair, and UV-induced mutagenesis.

Main Methods:

• Utilized a shuttle vector plasmid (pLSC) containing a poly(G) run in a tRNA gene as a mutagenic target.
• Compared mutation frequencies and types in mismatch repair-proficient (TK6) and deficient (MT1, hMSH6-deficient) human cell lines.
• Sequenced independent plasmid mutants to characterize alterations.
• Assessed the impact of UV treatment on mutagenesis in both cell lines and plasmid constructs.

Main Results:

• Spontaneous mutation frequencies were similar in proficient and deficient cell lines, but mutation classes differed significantly.
• Single base deletions within the poly(G) run were significantly more frequent in hMSH6-deficient cells (48%) compared to proficient cells (18%).
• UV treatment increased overall mutations primarily through base substitutions, with no significant increase in poly(G) run deletions.

Conclusions:

• Mononucleotide poly(G) runs promote single base deletion mutations.
• This mutagenic effect is exacerbated in cells deficient in hMSH6-mediated mismatch repair.
• The promotion of deletions by poly(G) runs is independent of UV-induced mutagenesis.