Nonhomologous end joining mechanisms conservation and relationship to illegitimate recomb

Nonhomologous end-joining: mechanisms, conservation and relationship to illegitimate recombination Thomas E. Wilson. in addition to mediating DNA end synapsis to prevent illegitimate mechanisms that govern whether a DSB is repaired by NHEJ or HR remain Key words: Dictyostelium, Double-strand break repair, Non-homologous end-joining, Homologous recombination stability and the relationship between these two pathways at. The synapsis factors of the S. cerevisiae non-homologous end-joining .. in protection of DNA ends from nucleolytic processing was proposed [80]. . mechanisms of NHEJ producing either insertions or deletions and that these RAD52, and related genes on illegitimate recombination in Saccharomyces cerevisiae.

HR in fact refers to several different processes, including gene conversion and alternative pathways called break-induced replication BIR and single strand annealing SSA.

A key-protein in HR is Rad52, which forms a heptameric ring in vitro 3that interacts with single stranded DNA and facilitates the strand invasion reaction 45. Rad52 is thought to act early during HR and strains lacking Rad52 are completely deficient for all types of HR in Saccharomyces cerevisiae.

Alternative end-joining mechanisms: a historical perspective

Another important protein is Rad51 similar to bacterial RecAwhich facilitates the strand exchange reaction between the damaged substrate and the undamaged template molecule 6. In contrast, the NHEJ pathway requires little or no homology and simply fuses two free DNA-ends, often generating small deletions and insertions. The heterodimer binds to the DSB protecting the free ends from extensive degradation and holds the DNA ends in an appropriate configuration for subsequent end-processing and ligation 8.

This complex consists of a DNA ligase, Dnl4 or Lig4 10 — 12 and Lif1 Ligase four interacting factor 1 13the latter being the yeast functional homologue of mammalian Xrcc4. Lif1 is required for the stability and full activity of Dnl4 Diploid cells can rely on the HR pathway to repair DSBs even in the G1 phase of the cell cycle, since these cells contain a homologous chromosome.

Despite the high degree of molecular conservation in NHEJ mechanisms between yeast and mammalian cells, there are some striking differences regarding the in vivo function.

Thus, inactivation of the NHEJ pathway would lead to an increase of homologous integration events upon introduction of a gene targeting cassette. This hypothesis was tested in mouse ES-cells, but NHEJ mutant cells did not have a more favorable gene targeting frequency Hence, IR between two genomic loci results in deletions, duplications, insertions or translocations.

In addition, overexpression of Top1 leads to increased IR, demonstrating that the nicking activity of Top1 promotes IR Similarly to mammalian cells, milk yeast NHEJ was capable of repairing blunt and noncohesive ends efficiently. Integration of a nonhomologous DNA molecule into the genome was fold more frequent than in S.

Alternative end-joining mechanisms: a historical perspective

We present evidence supporting a model in which IR takes place at spontaneous mitotic DSB and that these breaks occurred more frequently within promoter regions and rDNA. Obviously, the predominating DSB repair pathway in K. Our aim was to answer the question of whether P.

Using the sequence of the PsKU80 gene that we found in the P. The following yeast strains were used: The latter strain was constructed by integration of a reporter cassette next to the original ADH1 locus of the strain PJH The strain was used for the isolation of regulatory mutants in which the expression of ADH1 was prevented. In this study, we used the nomenclature proposed by Jeffrieswhere the major ADH-gene responsible for ethanol formation and consumption is designated as ADH1.

AJ ], a 2. AFAmpR and a bacterial origin of replication Hagedorn et al. It was constructed as follows: Minimal medium MM containing 1. Genomic yeast DNA was extracted as described by Johnston In case of cotransformation, restriction enzymes were added to the DNA directly before transformation. Yeast transformations were performed using the freezing method Dohmen et al. To obtain optimum competence, cells were frozen for only 1 h. Of each transformant clone, descendants ranging between and were tested.

After this restriction, a 1. The purified PCR product was used for transformation of P. Thus, the primers would produce 3. Results Untargeted integration mutagenesis Transformation efficiency using different vector types, stability of transformants Transformations of the recipient strain P. As documented by the high SD, the reproducibility of the single transformation experiments was low.

The highest yield was obtained with cells that had been deep frozen for only 1 h. The yield of transformants per number of cells increased with an increasing amount of vector DNA Fig. However, this effect was not very strong and never exceeded a twofold enhancement when compared with the efficiency without restriction enzyme.

Varying the amounts of EcoRI between 1 and 30 U per transformation had no additional enhancing effect on the transformation efficiency 2.