Transformation with Oligonucleotides Creating Clustered Changes in the Yeast Genome

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.

In Vivo Bypass of 8-oxodG

“…In order to use oligos to incorporate a segment of DNA, it was necessary to know the frequency of co-incorporation of nucleotides in a given oligo. Using oligos with markers spread throughout the length of the oligo (Oligo N, Figure 1), we determined that for an oligo of 40 nt in length, a central core of 10–15 nt was incorporated with a greater than 90% frequency [43]. Those results suggested that it was feasible to use oligos of that length for our experiments.…”

“…A potential problem was that the region surrounding the mutant base is highly conserved, constraining the location of any damaged base. We therefore created the mutant trp5-G148Cm gene (Figure 1) [43]. Because this mutant trp5-G148Cm gene is placed close to a dependable origin of replication, and is present in both orientations relative to the origin, we know which strand is replicated as leading and which as lagging and can reverse the replication strands by using a strain of opposite TRP5 orientation [42].…”

“…However, our prior experiments [43] as well as a number of preliminary experiments indicated that we needed a method to mark incorporation of bases on both sides of the damaged base in order to be sure that we were observing bypass, and not partial incorporation of the relevant region of the oligo. These goals were accomplished by transforming with Oligos G and GO (Figure 1).…”

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

“…Prior work (Rodriguez et al, 2012) implicated the Fen1-endonuclease in flap degradation at the 5′ end of ssODNs. We posited that this effect could be partially explained by truncated ssODNs arising from errors in DNA synthesis since ssODNs are synthesized 3′ to 5′.…”

Yeast Oligo-Mediated Genome Engineering (YOGE)

“…We found that variability in strand bias in the examined loci was consistent between strains and could not be solely explained by transcriptional effects (Figure 4) , which lend further mechanistic support for lagging strand incorporation of oligos at the DNA replication fork. (15, 21, 22)…”