DNA replication involves sophisticated tag-team polymerase activity. The primary lagging strand polymerases α and δ are involved in Okazaki fragment synthesis, with a handover occurring after the pol α-primase complex synthesizes about 10-30 nucleotides of RNA/DNA primer.

Surprisingly, while the pol α-primase complex only synthesizes about 1.5% of the genome, it is more error prone than the other major polymerases because it lacks proofreading ability. Previous studies indicate that pol δ may be able to extrinsically proofread errors made by pol α during the handover using its exonuclease. A double mutant of pol1-L868M pol3-5DV in Saccharomyces cerevisiae, containing a mutator form of pol α and an exonuclease deficient form of pol δ, exhibits high rates of indels in long homopolymer runs.

Here, we aim to determine if repair of indels by pol δ could serve as a marker for extrinsic proofreading. By transforming select yeast with construct DNA containing mNeonGreen, a drug resistance gene, and an A homopolymer run, indel mutations within the homopolymer run should result in a fluorescent signal.

Currently, we've been able to construct DNA containing the target genes and homopolymer track of A's. Remaining steps include successful transformation into wild-type, pol1-L868M single mutant, pol3-5DV single mutant, and double mutant yeast. Following successful development of the assay, we plan to screen mutant libraries of pol δ for other mutations that may impact extrinsic proofreading.