DNA Replication in Plants Origins of DNA Replication and Post-translational Histone Modifications During the DNA Synthesis Phase of Arabidopsis thaliana.

FRANCO-RUIZ, MARIANA. DNA Replication in Plants: Origins of DNA Replication and Post-translational Histone Modifications during the DNA Synthesis phase of Arabidopsis thaliana. (Under the direction of George C. Allen and William F. Thompson). Mapping origins of DNA replication on a genomic scale remains a major challenge in eukaryotic molecular genetics. Although a few maps exist, notably for the lower eukaryote Saccharomyces cerevisiae, it has proven difficult to map replication origins to unique loci in the complex genomes of higher eukaryotes. In large measure, this is because (in contrast to yeast and prokaryotes) higher eukaryotes seem to lack a clear consensus sequence for replication origins. However, all eukaryotes use a similar mechanism of replication, in which the pre-replicative complex binds a DNA sequence (origin), unwinds the DNA to allow the replication machinery to start producing newly synthesized DNA or nascent strands. Such similarities offer hope that genomic tools can be used to define origins of DNA replication in higher eukaryotes. The first phase of the work described here is an effort to map origins of DNA replication in Arabidopsis thaliana using three different approaches: enrichment of nascent strands labeled with bromo-deoxy-uridine (BrdU), enrichment of nascent strands by size selection and enrichment of nascent strands by the use of lambda exonuclease. DNA enriched by each procedure was mapped onto custom-designed high resolution microarrays representing Arabidopsis thaliana chromosome 4. The nascent strands obtained with BrdU were not reproducible. For the nascent strands obtained by size selection, we got 721 putative origins and with the use of lambda exonuclease we obtained 330 putative origins. The two later methods had 217 putative origins in common. Comparison of the two dataset with features for chromosome 4 (Genes, coding sequences, exons, transposable elements etc) showed that the two sets contain different features. Further analyses are required to identify true origins. A second phase of the project sought to describe the abundance and distribution of various post-translational modifications of histones H3 and H4, using both Western blot and immunolocalization techniques. Modified histones play an important role in transcription and according to recent studies also in replication due to their role in chromatin structure. We found out that histone 4 acetylated in the lysine 5 and 16, and histone 3 acetylated in the lysine 56 do not present changes during the cell cycle in our cell culture. We also observe that histone 3 dimethylated in lysine 9 and phosphorylated in serine 10, present very low abundance and showed signal in the nucleolus. The data provide a basis for a future analysis of how these epigenetic marks are transmitted during DNA replication and mitosis. DNA Replication in Plants Origins of DNA Replication and Post-translational Histone Modifications During the DNA Synthesis Phase of Arabidopsis thaliana by Mariana Franco Ruiz A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Horticultural Science Raleigh, North Carolina 2012