Figure 1.
Structure of the URA3 allele on S. cerevisiae chromosome V used for these experiments. The position of various oligonucleotide primers (numbers and half arrows) used for PCR and sequencing are shown. The ACT1 intron placed into URA3 is normally spliced, resulting in uracil prototrophy (Ura+) and sensitivity to the drug FOAS. After creating a DSB within the engineered ACT1 intron with HO endonuclease, cells either die, are repaired in a way allowing normal splicing, or are repaired in a way that prevents splicing. The latter situation leads to a phenotype of uracil auxotrophy (Ura−) and resistance to 5-FOA (FOAR).
Figure 2.
Measurements of NHEJ ability of non-essential DNA polymerases.
The efficiency of NHEJ of wild-type and polymerase defective mutants, rev3, pol4 and 3′ to 5′ exonuclease defective mutants, pol2-4, pol3-01, pol2-4 pol4, dnl4, and pol2-4 dnl4 were measured. (A) Frequencies of imprecise end joining. The survival frequency was evaluated by the ratio of YPGal/YPD. (B) Frequency of chromosomal rearrangement was evaluated by the ratio of FOAR/YPD. Each experiment was collected from at least four independent clones. *: P<0.05; **: P<0.01. (C) The sequence analysis of the imprecise end joining events from the WT and pol4 mutants. The structures of various imprecise end joining repairs were identified at the DSB site. Sequences of the HO cut site are shown, with the resulting 3′ overhanging terminal AACA shown on the cut site. Samples of independent joints were PCR amplified and sequenced. A total of 50 independent survivors on YPGal plates from WT and pol4 mutant were examined. Bases underlined indicate insertion or mutation. The numbers of inserted (+) and deleted (−) bases were denoted in brackets. N indicates the events of each repair pattern.
Table 1.
Repair patterns after a DSB at the URA3::ACT1 intron::HO cut site locus of non-essential DNA polymerase mutants.
Figure 3.
Measurements of NHEJ ability of essential DNA polymerases.
The NHEJ assay was performed for pol1, pol3, pol2-18 and pol2-18 pol4 at permissive (23°C) and semi-permissive (30°C) temperatures. (A) Frequencies of imprecise end joining. The survival frequency was evaluated by the ratio of YPGal/YPD. (B) Frequency of chromosomal rearrangement was evaluated by the ratio of FOAR/YPD. Each experiment was collected from at least five independent clones. Dark gray bars indicate 23°C incubation and light gray bars indicate 30°C incubation. *: P<0.05; **: P<0.01. (C) Survivors of pol2-18 or pol2-4 mutants from YPGal plates at 30°C were examined. Bases underlined indicate insertion or mutation. The numbers of inserted (+) and deleted (−) bases were denoted in brackets. N indicates the events of each repair pattern.
Table 2.
Repair patterns after a DSB at the URA3::ACT1 intron::HO cut site locus of essential DNA polymerase mutants.
Table 3.
Yeast strains used in this study.
Figure 4.
Proposed pathways for imprecise end joining of the HO endonuclease induced DSB at MATa.
The 4-bp 3′ overhanging sequence resulting from HO endonuclease cleavage is indicated. The two ends must be brought together into synapsis. Precise religation would lead to re-cutting. Possible end processing and subsequent alignment of complementary base pairs that would lead to insertions or deletions are shown. When 3′ overhangs mispair, Pol4 is required to fill in the gap [15],[16]. When 3′ flaps are generated by mis-alignment, the 3′ to 5′ exonuclease activity of Pol2 is the predominate exonuclease to remove the flaps.