Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Organelle genomes in plants and protozoans are replicated by a unique DNA polymerase family.
• Mutator versions of animal gamma DNA polymerases advanced mitochondrial genome research, but similar tools are lacking for plant organelles.

Purpose of the Study:

• To create and characterize error-prone tobacco organelle DNA polymerases.
• To investigate methods for increasing mutation rates in plant organelle DNA replication.

Main Methods:

• A novel positive selection method using the phage lambda cI repressor gene was employed.
• The fidelity of tobacco organelle DNA polymerases was tested by altering exonuclease function and polymerisation domains.

Main Results:

• Ablating the 3'-5' exonuclease function modestly increased the error rate (5-8 fold).
• Combining exonuclease deficiency with polymerisation domain substitution yielded a 140-fold error rate increase, comparable to animal mutator polymerases.
• Mutations primarily consisted of single base substitutions, including frequent A:A mispairings.

Conclusions:

• Engineered tobacco organelle DNA polymerases exhibit significantly elevated error rates.
• These high-fidelity mutator enzymes are suitable for increasing mutation rates in plant chloroplasts and mitochondria.