Deletions initiated by the vaccinia virus TopIB protein in yeast.

The type IB topoisomerase of budding yeast (yTop1) generates small deletions in tandem repeats through a sequential cleavage mechanism and larger deletions with random endpoints through the nonhomologous end-joining (NHEJ) pathway. Vaccinia virus Top1 (vTop1) is a minimized version of the eukaryal TopIB enzymes and uniquely has a strong consensus cleavage sequence: the pentanucleotide (T/C)CCTTp↓. To define the relationship between the position of TopIB cleavage and mutagenic outcomes, we expressed vTop1 in yeast top1Δ strains containing reporter constructs with a single CCCTT site, tandem CCCTT sites, or CCCTT sites separated by 42 bp. vTop1 cleavage at a single CCCTT site was associated with small, NHEJ-dependent deletions. As observed with yTop1, vTop1 generated 5-bp deletions at tandem CCCTT sites. In contrast to yTop1-initiated deletions, however, 5-bp deletions associated with vTop1 expression were not affected by the level of ribonucleotides in genomic DNA. vTop1 expression was associated with a 47-bp deletion when CCCTT sites were separated by 42 bp. Unlike yTop1-initiated large deletions, the vTop1-mediated 47-bp deletion did not require NHEJ, consistent with a model in which re-ligation of enzyme-associated double-strand breaks is catalyzed by vTop1.

Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.

A targeted double-strand break introduced into the genome of Saccharomyces cerevisiae is repaired by the relatively error-prone nonhomologous end joining (NHEJ) pathway when homologous recombination is not an option. A zinc finger nuclease cleavage site was inserted out-of-frame into the LYS2 locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5' overhangs. Repair events that destroyed the cleavage site were identified either as Lys+ colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+ events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol ζ and Pol η. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required translesion synthesis polymerases as well as the exonuclease activity of the replicative Pol δ DNA polymerase. Survivors were equally split between NHEJ events and 1.2 or 11.7 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3' tails. Finally, NHEJ was more efficient in nongrowing than in growing cells and was most efficient in G0 cells. These studies provide novel insights into the flexibility and complexity of error-prone DSB repair in yeast.

Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.

A targeted double-strand break introduced into the genome of Saccharomyces cerevisiae is repaired by the relatively error-prone nonhomologous-end joining (NHEJ) pathway when homologous recombination is not an option. A ZFN cleavage site was inserted out-of-frame into the LYS2 locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5' overhangs. Repair events that destroyed the cleavage site were identified either as Lys + colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys + events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol σ and Pol 11. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required TLS polymerases as well as the exonuclease activity of the replicative Pol DNA polymerase. Survivors were equally split between NHEJ events and 1 kb or 11 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3' tails. Finally, NHEJ was more efficient in non-growing than in growing cells and was most efficient in G0 cells. These studies provide novel insight into the flexibility and complexity of error-prone DSB repair in yeast.

Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast.

Transcription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.

Mutagenic repair of a ZFN-induced double-strand break in yeast: Effects of cleavage site sequence and spacer size.

Double-strand breaks are repaired by error-free homologous recombination or by relatively error-prone pathways that directly join broken ends. Both types of repair have been extensively studied in Saccharomyces cerevisiae using enzymes HO or I-SceI, which create breaks with 4-nt 3' overhangs. In the current study, a galactose-regulated zinc-finger nuclease (ZFN) designed to cleave the Drosophila rosy locus was used to generate breaks with 4-nt 5' overhangs at out-of-frame cleavage sites inserted into the yeast LYS2 gene. Mutagenic repair was examined following selection of prototrophs on lysine-deficient medium containing galactose or surviving colonies on galactose-containing rich medium. Following cleavage of the original rosy spacer (ACGAAT), most Lys+ colonies contained 1- or 4-bp insertions at the cleavage site while most survivors had either a 2-bp insertion or a large deletion. Small insertions reflected nonhomologous end joining (NHEJ) and large deletions were the product of microhomology-mediated end joining (MMEJ). Changing the original ACGAAT spacer to either AGCAAT, ACGCGT or CTATTA altered the molecular features of NHEJ events as well as their frequency relative to MMEJ. Altering the optimal 6-bp spacer size between the zinc-finger protein binding sites to 5 bp or 7 bp eliminated the effect of continuous ZFN expression on survival, but Lys+ prototrophs were still generated. Analysis of Lys+ revertants after cleavage of the 5-bp spacer indicated that both the position and spacing of ZFN-generated nicks were variable. Results provide insight into effects of overhang sequence on mutagenic outcomes and demonstrate ZFN cleavage of 5- or 7-bp spacers in vivo.

Trapped topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast.

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5' end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) the product of which generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that overexpress the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.

Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.

Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3' overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3' end affects recombination outcomes and whether the presence of a 3' vs. 5' overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3' end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3' vs. 5' overhangs differs, we compared the well-studied 3' overhang created by I-SceI to a 5' overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.