Genome wide studies have uncovered the existence of large-scale copy number variation (CNV) in the human genome. The human genome of different individuals was initially estimated to be 99.9% similar, but population studies on CNV have revealed that it is 12-16% copy number variable. Abnormal genomic CNVs are frequently found in subtelomeres of patients with mental retardation (MR) and other neurological disorders. Rearrangements of chromosome subtelomeric regions represent a high proportion of cytogenetic abnormalities and account for approximately 30% of pathogenic CNVs. Although DNA double strand breaks (DSBs) are implicated as a major factor in chromosomal rearrangements, the causes of chromosome breakage in subtelomeric regions have not been elucidated. But due to the presence of repetitive sequences in subtelomeres, we hypothesized that chromosomal rearrangements in these regions are not stochastic but driven by specific sequence motifs. In a collaborative effort with Dr. Rudd (Department of human genetics at Emory University), we characterized subtelomeric breakpoints on different chromosome ends in search of common motifs that cause double-strand breaks. Using a yeast-based gross chromosomal rearrangement (GCR) system, we have identified a subtelomeric breakage motif from chromosome 2 (2q SBM) with a GCR rate that is 340 fold higher than background levels. To determine if the fragility of 2q SBM was driven by the formation of secondary structures, the helicase activities of Sgs1 and Pif1 were disrupted. These helicases have been shown to destabilize DNA secondary structures such as G-quadruplex structures. Disruption of these helicases augmented chromosomal rearrangements induced by 2q SBM, indicating that these helicases are required for maintenance of this sequence. We also donwregulated replication fork components to determine if 2q SBM was imposing any problems to the replication fork machinery. Downregulation of replication fork components increased chromosomal rearrangements, indicating that intact replication fork was a critical determinant of 2q SBM fragility. Using a yeast-based functional assay, these experiments have linked human subtelomeric repetitive sequences to chromosomal breakage that could give rise to human CNV in subtelomeric regions.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/41119 |
| Date | 24 May 2011 |
| Creators | Khuzwayo, Sabelo Lethukuthula |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Thesis |
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