The NHEJ pathway in yeast is very similar to that of mammalian cells but with important differences. Whereas budding yeast has homologs of the two mammalian Ku subunits, it appears to lack a DNA-PKcs homolog [19
]. Also, there does not seem to be a clear homolog of Artemis [18
]. Yeast cells do have Mre11p/Rad50p/Xrs2p and Lig4p/Lif1p complexes, however, and these are functional homologs of the mammalian MRE11/RAD50/NBS1 and ligase IV/XRCC4 complexes, respectively [19
]. In contrast to higher eukaryotes, budding yeast relies more on homologous recombination than on NHEJ to deal with DSBs. Consequently, deletion of yeast NHEJ genes in the presence of a functional homologous recombination pathway does not lead to a readily measurable change in sensitivity to DNA-damaging agents [21
]. This 'masking' of defects in NHEJ by homologous recombination activity means that it is not easy to perform a conventional genetic screen in yeast to identify new NHEJ factors.
Boeke and colleagues [8
] have recently used an elegant system to overcome this problem, taking advantage of a special collection of yeast deletion mutants. In this collection, over 5,800 of the estimated 6,000 yeast open reading frames (ORFs) have been systematically disrupted. In each mutant, the complete ORF has been replaced by a KanMX
cassette, which results in resistance to the antibiotic G418. Furthermore, each cassette is distinctly marked by a special 'barcode' consisting of two 20-nucleotide sequences flanking the KanMX
gene. These are referred to as UPTAGs and DOWNTAGs and are themselves flanked by universal priming sites. The two tags can be used as hybridization probes to detect the presence of each mutant in a mixed population of mutants (Figure ) [22
Figure 1 The microarray screen performed by Ooi et al. . The S. cerevisiae-Escherichia coli shuttle plasmids pRS416 are linearized with the restriction endonuclease EcoRI. The mutant pools are transformed in parallel with circular and linearized pRS416 plasmids (more ...)
In the paper by Ooi et al.
], this technology is used to identify new factors in the NHEJ pathway. They took advantage of an assay in which yeast has to repair a DSB using NHEJ, despite the presence of a functional homologous recombination pathway. Thus, mutations in genes involved in NHEJ result in a measurable defect in DNA repair and the aforementioned 'masking' problem is avoided. The assay is an in vivo
plasmid repair assay, in which a DSB is generated within a region of a plasmid that is not homologous to chromosomal sequences; thus homologous recombination cannot be used to repair this DSB. Yeast cells transformed with this plasmid in either linearized or supercoiled form are grown and counted in parallel (to account for differences in transformation efficiencies) on selective medium. The linearized plasmids can be maintained in yeast only after they have been recircularized and ligated, so, the efficiency of transformation with linearized plasmid, normalized to that of super-coiled plasmid, provides a quantitative readout of DSB repair via the NHEJ pathway.
Ooi et al.
carried out this plasmid repair assay on pools of mutant haploid and homozygous diploid yeasts. Separate green- and red-labeled probes, derived from the linear and circular plasmid transformations, respectively, were hybridized to oligonucleotide arrays (Figure ; further details of the method are in the figure legend). NHEJ defective mutants can bind only one of the probes - the red one - and so produce a red signal while the rest of the array is yellow. Because the barcodes are unique to each mutant, the NHEJ mutants could be easily identified. Indeed, in this way Ooi et al.
] identified most of the known NHEJ genes, providing a clear 'proof of principle' for their method. A number of additional genes were shown to be important for NHEJ, but, for most of these ORFs, it is not immediately evident how they might function in the NHEJ pathway. One ORF discovered in this way, however, designated as YLR265C or NEJ1,
was known to code for a protein previously shown to interact with the Lif1p component of the Lig4 complex in a high-throughput two-hybrid study [23
]. Ooi et al.
] show in their paper that deletion of this ORF results in a defect in NHEJ and confirm that Nej1p interacts with the amino terminus of Lif1p in a two-hybrid assay.
Although Ooi et al.
] do not suggest a mechanism of action for Nej1p, other groups who independently identified NEJ1
go some way towards providing a possible mechanism of action [24
]. It seems that Nej1p is involved in regulation of NHEJ in a cell-type-dependent manner. Thus, in the diploid state, where a haploid yeast cell can potentially use error-free homologous recombination at all stages of the cell cycle, the NHEJ machinery is down-regulated through suppression of Nej1p expression. This is achieved by the Mata
1-Matα2 transcriptional repressor, which is expressed only in diploid cells and switches off haploid-specific genes [27
]. It turns out that NEJ1
is one of the targets of Mata
1-Matα2 suppression [24
]. When NEJ1
expression is repressed in diploid cells, this appears to result in the loss of nuclear localization of Lig4p [25
]. In this way, budding yeast has evolved an elegant system to favor the repair of DSBs by homologous recombination in diploid cells by repressing the expression of a subunit of the DNA Lig4 complex (Figure ). Given that NHEJ plays a particularly crucial role during G1 phase of a haploid cell cycle, when there are no homologous sequences available [28
], it will be interesting to see whether Nej1p is also regulated during different stages of the cell cycle.
Figure 2 A possible mechanism for the role of Nej1p in NHEJ. In diploid cells, expression of the Mata1-Matα2 repressor (red circles) results in transcriptional repression of NEJ1. This, in turn, causes a loss of the nuclear localization of Lif1p (yellow), (more ...)