Cis and trans-acting effects on a mutational hotspot involving a replication template switch

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• The thyA gene in Escherichia coli harbors a natural mutational hotspot responsible for over half of inactivating mutations.
• This hotspot, a T to A transversion at base 131, is located within a quasi-palindromic sequence and is selectable by trimethoprim resistance.

Purpose of the Study:

• To elucidate the mechanism of mutagenesis at the thyA131 hotspot.
• To investigate the cis and trans-acting factors influencing hotspot activity in its natural chromosomal context.

Main Methods:

• Analysis of mutations within the quasi-palindromic sequence and surrounding DNA.
• Assessment of hotspot activity under various genetic conditions, including alterations in DNA pairing, operon inversion, and deficiencies in DNA repair polymerases and exonucleases.

Main Results:

• Mutagenesis is confirmed to proceed via a template-switch mechanism, with strengthened base-pairing stimulating and weakened pairing reducing hotspot activity.
• Co-mutation of multiple sites within the quasipalindrome supports the templated DNA synthesis model.
• Hotspot mutagenesis occurs during both leading and lagging-strand synthesis and is independent of the PriA replication restart factor.
• Loss of SOS-induced DNA polymerases (PolII, PolIV, PolV) significantly increased mutation rates, revealing an antimutagenic role.
• Deficiency in ExoI and ExoVII exonucleases dramatically stimulated hotspot mutagenesis, indicating their role in aborting premutagenic events.

Conclusions:

• The thyA131 hotspot is a robust example of template-switching during chromosomal replication.
• SOS-induced DNA polymerases and major 3' single-strand DNA exonucleases play critical, redundant roles in suppressing hotspot mutagenesis.