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Saccharomyces cerevisiae mating-type switching is initiated by a double-strand break (DSB) at MATa, leaving one cut end perfectly homologous to the HMLα donor, while the second end must be processed to remove a nonhomologous tail before completing repair by gene conversion (GC). When homology at the matched end is ≤150 bp, efficient repair depends on the Recombination Enhancer, which tethers HMLα near the DSB. Thus homology shorter than an apparent “minimum efficient processing segment” can be rescued by tethering the donor near the break. When homology at the second end is ≤150 bp, second-end capture becomes inefficient and repair shifts from GC to break-induced replication (BIR). But when pol32 or pif1 mutants block BIR, GC increases three-fold, indicating that the steps blocked by these mutations are reversible. With short second-end homology, absence of the RecQ helicase Sgs1 promotes synthesis-dependent strand annealing whereas deletion of the FANCM-related Mph1 helicase promotes BIR.

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We have previously shown that a recombination execution checkpoint (REC) regulates the choice of the homologous recombination pathway used to repair a given DNA double-strand break (DSB) based on the homology status of the DSB ends. If the two DSB ends are synapsed with closely-positioned and correctly-oriented homologous donors, repair proceeds rapidly by the gene conversion (GC) pathway. If, however, homology to only one of the ends is present, or if homologies to the two ends are situated far away from each other or in the wrong orientation, REC blocks the rapid initiation of new DNA synthesis from the synapsed end(s) and repair is carried out by the break-induced replication (BIR) machinery after a long pause. Here we report that the simultaneous deletion of two 39/59 helicases, Sgs1 and Mph1, largely abolishes the REC-mediated lag normally observed during the repair of large gaps and BIR substrates, which now get repaired nearly as rapidly and efficiently as GC substrates. Deletion of SGS1 and MPH1 also produces a nearly additive increase in the efficiency of both BIR and long gap repair; this increase is epistatic to that seen upon Rad51 overexpression. However, Rad51 overexpression fails to mimic the acceleration in repair kinetics that is produced by sgs1D mph1D double deletion. While NHEJ involves simple religation of the DSB ends with little or no homology, HR requires the presence of intact homologous sequences to serve as a template for repair. During HR-mediated repair, the DSB ends are resected to produce 39-ended single-stranded DNA tails, which get coated with the Rad51 recombinase protein to form Rad51 nucleoprotein filaments. These Rad51 filaments then search for and strand invade homologous sequences to form a three-strand displacement loop (D-loop), which is followed by extension of the invading strand by new DNA synthesis using the paired homologous sequence as a template. When both DSB ends share homology with a sister chromatid, a homologous chromosome or an ectopically placed donor; repair occurs by gene conversion (GC), resulting in the synthesis of a short patch of new DNA at the recipient locus using the homologous donor as a template. New DNA synthesis is initiated within 30 min after strand invasion (White and Haber 1990;Sugawara et al. 2003;Hicks et al. 2011) and does not require components of the lagging strand DNAsynthesis machinery such as Pola and primase (Wang et al. 2004). In mitotic cells of budding yeast, GC proceeds primarily by the synthesis-dependent strand annealing (SDSA) mechanism, in which the newly-synthesized strands dissociate from the donor template and are returned to the recipient locus. GC is thus distinct from normal semiconservative

Ferritin, a symmetrical 24-subunit heteropolymer composed of heavy and light chains, is the primary iron-storage molecule in bacteria, plants and animals. We used a genetically engineered strain of the model organism Drosophila melanogaster which expresses a GFP (green fluorescent protein)-tagged ferritin 1 heavy chain homologue from its native chromosomal locus and incorporated it into endogenous functional ferritin, enabling in vivo visualization of the protein and permitting easy assessment of ferritin status following environmental or genetic perturbations. Random mutagenesis was induced, and individual mutagenized chromosomes were recovered by classic crossing schemes involving phenotypical markers and balancer chromosomes. In wild-type larvae, ferritin is predominantly localized in the brain, in regions of the intestine, in wreath cells and in pericardial cells. A pilot genetic screen revealed a mutant fruitfly strain expressing GFP-ferritin in the anal pads, a pair of organs located ventrally in the posterior end of the fruitfly larva, possibly involved in ion absorption and osmoregulation, which are normally devoid of ferritin. Our continuing genetic screen could reveal transcription factors involved in ferritin regulation and novel proteins important in iron metabolism, hopefully with conserved functions in evolution.