Carlos Flores

The Drosophila Mre11/Rad50 Complex Is Required to Prevent Both Telomeric Fusion and Chromosome Breakage

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.

Differential usage of alternative pathways of double-strand break repair in Drosophila.

Double-strand DNA breaks can be repaired by any of several alternative mechanisms that differ greatly in the nature of the final repaired products. We used a reporter construct, designated “Repair reporter 3” (Rr3), to measure the relative usage of these pathways in Drosophila germ cells. The method works by creating a double-strand break at a specific location such that expression of the red fluorescent protein, DsRed, in the next generation can be used to infer the frequency at which each pathway was used. A key feature of this approach is that most data come from phenotypic scoring, thus allowing large sample sizes and considerable precision in measurements. Specifically, we measured the proportion of breaks repaired by (1) conversion repair, (2) nonhomologous end joining (NHEJ), or (3) single-strand annealing (SSA). For conversion repair, the frequency of mitotic crossing over in the germ line indicates the relative prevalence of repair by double Holliday junction (DHJ) formation vs. the synthesis-dependent strand annealing (SDSA) pathway. We used this method to show that breaks occurring early in germ-line development were much more frequently repaired via single-strand annealing and much less likely to be repaired by end joining compared with identical breaks occurring later in development. Conversion repair was relatively rare when breaks were made either very early or very late in development, but was much more frequent in between. Significantly, the changes in relative usage occurred in a compensatory fashion, such that an increase in one pathway was accompanied by decreases in others. This negative correlation is interpreted to mean that the pathways for double-strand break repair compete with each other to handle a given breakage event.

A Third Link connecting Aging with Double Strand Break Repair

Until recently, the connection between aging and DNA repair has rested on two classes of observation. First, DNA damage and unrepaired double-strand breaks (DSBs) accumulate with age. Second, several defects in DNA repair genes are associated with early onset of age-related diseases and other signs of premature aging. Now, a third link has emerged: The mechanisms by which cells repair DSB damage can change dramatically with age, shifting from simpler end-joining processes in younger organisms to homologous mechanisms in which missing genetic information is restored through use of a template. So far this third link between aging and DNA repair has only been observed in a small number of experimental systems, and cannot yet claim the generality of the other two. Here we review the evidence for this phenomenon and present new data testing models for the underlying causes. If the generality of age-related changes in DSB repair pathway usage can be established, it will provide a new insight into the underlying molecular basis of aging and how evolution has shaped these processes.

The Gos28 SNARE protein mediates intra-Golgi transport of rhodopsin and is required for photoreceptor survival.

Background: The Golgi SNARE, Gos28, plays important roles in vesicular transport during protein trafficking. Results: Mutations in gos28 lead to defective rhodopsin trafficking and retinal degeneration, which is rescued by human Gos28 expressed in transgenic flies. Conclusion: Drosophila Gos28 functions as a t-SNARE in medial- to trans-Golgi transport of rhodopsin during its biosynthesis. Significance: Gos28 represents a novel locus in neurodegeneration. SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28s SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.

Jumping around the Topic of Mobile Genetic Elements

Behe Michael J., Darwins Black Box, 1996, Free Press, New York, 289,

Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer

25.00Bell, R.M., Exton, J.H., Prescott, S.M., Handbook of Lipid Research, 1996, Plenum Press, New York, 316,

Microsatellite instability in Drosophila spellchecker1 (MutS homolog) mutants

89.50Birren, B., Lai, E., Nonmammalian Genomic Analysis, A Practical Guide, , 1996, Academic Press, San Diego, California, 353,

Age-dependent usage of double-strand-break repair pathways.

39.95Biswas, B.B., Biswas, S., Subcellular Biochemistry, Vol 26, myo-Inositol Phosphates, Phosphoinositides/ and Signal Transduction, 1996, Plenum Press, New York, 421,

Multiple-Pathway Analysis of Double-Strand Break Repair Mutations in Drosophila

125.00Boyce, A.J., Mascie-Taylor, C.G.N., Molecular Biology and Human Diversity, 1996, Cambridge University Press, New York, 305,

Efficient repair of DNA breaks in Drosophila: evidence for single-strand annealing and competition with other repair pathways.

64.95Brabec, V., Walz, D., Milazzo, G., Experimental Techniques in Bio Electrochemistry, 1996, Birkhauser, Boston, Massachusetts, 558,