Re-replication is the aberrant duplication of genomic DNA more than once per cell cycle. Re-replication causes DNA double strand breaks and can lead to genomic instability and oncogenesis (Hook et al, 2007). Previous results from the lab have indicated that over-expression of the origin binding protein Cdt1 (double parked or Dup in flies) is sufficient to induce re-replication in a variety of developing Drosophila tissues (Thomer et al, 2004). An Array Comparative Genomic Hybridization (aCGH) comparing normal and re-replicating cells from brains and discs done in collaboration with the MacAlpine lab suggests that the largely heterochromatic fourth chromosome re-replicates preferentially. By using larval salivary gland nuclei as a low resolution genomic array, I have shown that alpha heterochromatin re-replicates preferentially. I examined what feature of heterochromatin makes it re-replication sensitive.
I have shown that Painting Of Fourth, a protein that is unique to the fourth chromosome displays a larger domain of labeling in re-replicating nuclei. The AT rich simple satellite 1.672-38 sequence unique to the fourth in females also appears larger. Most importantly, I found that the [(AATAT)n]/1.672-38 sequence re-replicates to higher copy numbers than the closely related [(AATAC)n]/1.672-181 sequence in re-replicating nuclei, in both fluorescence and radioactivity quantification assays. This indicates a possible sequence bias to re-replicating loci in Drosophila melanogaster.
In other work, I have shown that over-expression of Dup in larval salivary glands causes short term inhibition of incorporation of BrDU, a S phase marker. Pattern of labeling is focal in experimental sample, not band like as in control, suggesting fork collapse. Inhibition of BrDU incorporation is enhanced when the over-expression occurs in a p53 mutant background, indicating a hitherto unknown role for p53 in suppression of fork restart in Drosophila.
Further investigations may lead to important insights into the relationship between chromosome structure and origin activity during normal and aberrant DNA replication, and allow for prediction of fragile sites resulting from re-replication damage.
|Advisor:||Calvi, Brian R.|
|Commitee:||Michaels, Scott D., Walczak, Claire E.|
|School Location:||United States -- Indiana|
|Source:||MAI 51/04M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Genetics|
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