Telomeres consist of G-rich tandem repeats and their associated protein. Telomere proteins regulate the length of the telomeric DNA tract and protect ends from double-strand break (DSB) repair mechanisms. Proper telomere length is maintained by equilibrium between processes that shorten the telomere tract and those that elongate it. Telomere extension is accomplished primarily by the telomerase enzyme. Because telomerase can add telomere repeats to DSBs, it is possible to "heal" broken chromosomes by de novo telomere formation (DNTF). However, in most cases other DNA damage repair mechanisms predominate and DNTF is not observed. Maintaining telomere integrity is critical to preventing chromosome end fusions, but DNTF at chromosome breaks could lead to deletions and more complex rearrangements. In many cancer cells, chromosome deletions and other rearrangements involve DNTF. Although the factors required for DNTF are unknown, the presence of telomere repeat arrays increases the likelihood of DNTF at nearby DSBs. This study will use the model organism Arabidopsis thaliana to study the process of DNTF and determine factors that influence its efficiency. I will pursue four aims that exploit the genetic tractability and extensive mutant collections available in Arabidopsis. The first two aims will focus on developing a system to produce and detect DNTF events in Arabidopsis cells. For Aim 1, I will transform cells with a construct containing a telomere repeat array and assay for chromosome truncation resulting from DNTF. For Aim 2, I will express the l-Scel restriction enzyme in planta to cleave chromosomes at a specific transgenic loci. DSB repair by DNTF will be detected by loss of distal marker genes. In my third aim, I will test the effects of telomere repeat arrays of various sizes, positions and orientations on the efficiency of DNTF. In the final aim, the assay developed in the previous aims will be used to compare the frequency of DNTF among different genetic backgrounds. The proposed experiments test the role of telomere repeat arrays in predisposing a chromosome region to DNTF. Furthermore, the genetic requirements of DNTF will be tested. These experiments will provide insight into how endogenous telomeres are maintained, the process of DNTF, and the factors that foster DNTF. 2. Telomeres, the ends of chromosomes, are specialized structures required to prevent chromosome rearrangement. Such rearrangements lead to genome instability and are common in cancer cells. This study looks at how these structures form and tests which genes are required for their formation. [unreadable] [unreadable] [unreadable]