Transformation with Oligonucleotides Creating Clustered Changes in the Yeast Genome

Gina P Rodriguez,Gray F Crouse,Joseph B Song,David T Kirkpatrick

doi:10.1371/journal.pone.0042905

Abstract

We have studied single-strand oligonucleotide (oligo) transformation of yeast by using 40-nt long oligos that create multiple base changes to the yeast genome spread throughout the length of the oligos, making it possible to measure the portions of an oligo that are incorporated during transformation. Although the transformation process is greatly inhibited by DNA mismatch repair (MMR), the pattern of incorporation is essentially the same in the presence or absence of MMR, whether the oligo anneals to the leading or lagging strand of DNA replication, or whether phosphorothioate linkages are used at either end. A central core of approximately 15 nt is incorporated with a frequency of >90%; the ends are incorporated with a lower frequency, and loss of the two ends appears to be by different mechanisms. Bases that are 5–10 nt from the 5′ end are generally lost with a frequency of >95%, likely through a process involving flap excision. On the 3′ end, bases 5–10 nt from the 3′ end are lost about 1/3 of the time. These results indicate that oligos can be used to create multiple simultaneous changes to the yeast genome, even in the presence of MMR.

Highlights

It was first demonstrated in the Sherman lab that singlestranded oligonucleotides could be introduced into yeast cells and create permanent changes in the genome, the mechanism of that transformation was not understood [1,2,3]
Measuring incorporation of multiply-marked oligos Even under optimal conditions in the absence of mismatch repair (MMR), the frequency of oligo transformation is so low that one needs to be able to select for those cells that have successfully incorporated the oligo
If oligos serve as primers for replication, one might have expected them to transform only when annealed to the lagging strand of replication, which is replicated discontinuously, and not to the leading strand which is presumably replicated in a continuous fashion

Summary

Introduction

It was first demonstrated in the Sherman lab that singlestranded oligonucleotides (oligos) could be introduced into yeast cells and create permanent changes in the genome, the mechanism of that transformation was not understood [1,2,3]. Later work in both E. coli and yeast, in which events can be determined in relation to known origins of replication, has shown that transformation is more efficient when oligos anneal to the lagging strand [4,5]. Because the function of MMR is to remove newly replicated DNA or invading DNA that would create mismatches with the existing DNA, one might have expected MMR to interfere with the process of oligo transformation

Methods

Results

Conclusion