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School of Biological Sciences

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search.filters.applied.f.advisor: Lobachev, Kirill × Start date: 2000 × search.filters.applied.f.isOrgUnitOfPublicationTitle: College of Sciences ×

Publication Search Results

Now showing 1 - 5 of 5
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Multiple roles of the telomeric CST complex in preserving genome stability at sub-telomeric and difficult-to-replicate chromosomal regions

2022-05-20 , Guo, Wenying

The CST is an essential multiprotein complex composed of Cdc13, Stn1, and Ten1 in budding yeast and Ctc1, Stn1, and Ten1 in vertebrates. The complex is involved in telomere maintenance which is critical to genome stability. The complex binds to the telomeric single-stranded (ss) DNA overhangs and ensures proper telomere capping. Recent evidence suggests that the mammalian CST performs non-telomeric functions during replication stress. In my work, using budding yeast, I investigated how CST deficiency can affect genome instability at subtelomeric and non-telomeric regions. In the first part, I used the CDC13 defective allele I identified, cdc13-707fs. I found that upon Cdc13 deficiency, stretches of ssDNA can reach up to 50 kb. I further characterized the consequences of ssDNA exposure and found that exposed ssDNA is prone to hyper-mutability mediated by the translesion polymerase ζ during restoration synthesis. I determined the mode of restoration synthesis and found that it proceeds from the telomere towards the centromere. In the second part, I found that ssDNA generated as a result of telomere uncapping and 5’ → 3’ resection favors the cis interaction between inverted repeats (IR) located 43 kb away from the telomere. Using a set of different inverted repeats, I showed that the parameters that are known to constrain their fragility, namely divergency and long spacers, are not effective in cdc13-707fs. Importantly, I showed that the IR-mediated chromosomal rearrangements upon CST deficiency do not result from an increase in DNA breakage but rather from a DNA fold-back mediated by the annealing of the ssDNA followed by dicentric chromosome formation, its breakage in anaphase and subsequent repair events. I characterized the genetic dependency of IR-mediated fragility in cdc13-707fs and found that the ssDNA annealing proteins Rad52 and Rad59, as well as the structure-specific nucleases Slx4 and Rad1, are required for intrastrand interaction and inverted dicentric dimer formation. In the third part, I explored the non-telomeric functions of CST, which has never been studied in budding yeast. I found that CST deficiency leads to an increase in chromosomal breakage and recombination at inverted repeats located 343 kb away from the telomere. This suggests that the CST complex plays an important role at difficult-to-replicate chromosomal regions. These findings in yeast have implications for understanding mechanisms that shape eukaryotic genomes during evolution and development and mechanisms of hereditary diseases and tumorigenesis.

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Genome instability induced by triplex forming mirror repeats in S.cerevisiae

2009-04-07 , Kim, Hyun-Min

The main goal of this research is to understand molecular mechanisms of GAA/TTC-associated genetic instability in a model eukaryotic organism, S. cerevisiae. We demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin and to block replication fork progression. MutSbeta complex and endonuclease activity of MutLalpha play an important role in facilitation of fragility. In addition to GAA/TTC triplex forming repeats, non-GAA polypurine polypyrimidine mirror repeats that are prone to the formation of similar structures were found to be hotspots for rearrangements in humans and other model organisms. These include H-DNA forming sequences located in the major breakpoint cluster region at BCL2, intron 21 of PKD1, and promoter region of C-MYC. Lastly, we have investigated the effect of the triplex-binding small molecules, azacyanines, on GAA-mediated fragility using the chromosomal arm loss assay. We have found that in vivo, azacyanines stimulate (GAA/TTC)-mediated arm loss in a dose dependent manner in actively dividing cells. Azacyanines treatment enhances the GAA-induced replication arrest. We discovered that also, azacyanines at concentrations that induce fragility also inhibit cell growth. Over 60% of yeast cells are arrested at G2/M stage of the cell cycle. This implies an activation of DNA-damage checkpoint response.

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Understanding the mechanisms underlying DSB repair-induced mutagenesis at distant loci in yeast

2014-04-04 , Saini, Natalie

Increased mutagenesis is a hallmark of cancers. On the other hand, this can trigger the generation of polymorphisms and lead to evolution. Lately, it has become clear that one of the major sources of increased mutation rates in the genome is chromosomal break formation and repair. A variety of factors can contribute to the generation of breaks in the genome. A paradoxical source of breaks is the sequence composition of the genomic DNA itself. Eukaryotic and prokaryotic genomes contain sequence motifs capable of adopting secondary structures often found to be potent inducers of double strand breaks culminating into rearrangements. These regions are therefore termed fragile sequence motifs. Here, we demonstrate that in addition to being responsible for triggering chromosomal rearrangements, inverted repeats and GAA/TTC repeats are also potent sources of mutagenesis. Repeat-induced mutagenesis extends up to 8 kb on either side of the break point. Remarkably, error-prone repair of the break by Polζ reconstitutes the repeats making them a long term source of mutagenesis. Despite its negative connotations for genome stability, the mechanisms underlying the unstable nature of double strand break repair pathways are not known. Previous studies have demonstrated that break induced replication (BIR), a mechanism employed to repair broken chromosomes with only one repairable end, is highly mutagenic, undergoes frequent template switching and often yields half-crossovers. In the work presented here, we show that the instabilities inherent to BIR can be attributed to its unusual mode of synthesis. We determined that BIR proceeds via a migrating bubble with long stretches of single-stranded DNA and culminates with conservative inheritance of the newly synthesized DNA. We propose that the mechanisms described here might be important for generation of repair-associated mutagenesis in higher organisms. Secondary structure forming repeats like inverted repeats have been found to be enriched in cancer cells. These motifs often constitute chromosomal rearrangement hot-spots and demonstrate the phenomenon of kataegis. This study provides a mechanistic insight into how such breakage-prone motifs contribute to hypermutability of cancer genomes.

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Inverted repeats as a source of eukaryotic genome instability

2008-07-08 , Narayanan, Vidhya

Chromosomal rearrangements play a major role in the evolution of eukaryotic genomes. Genomic aberrations are also a hallmark of many tumors and are associated with a number of hereditary diseases in humans. The presence of repetitive sequences that can adopt non-canonical DNA structures is one of the factors which can predispose chromosomal regions where they reside to instability. Palindromic sequences (inverted repeats with or without a unique sequence between them) that can adopt hairpin or cruciform structures are frequently found in regions that are prone for gross chromosomal rearrangements (GCRs) in somatic and germ cells in different organisms. Direct physical evidence was obtained that double-strand breaks (DSBs) occur at the location of long inverted repeats, a triggering event for the genomic instability. However, the mechanisms by which palindromic sequences lead to chromosomal fragility are largely unknown. The overall goal of this research is to elucidate the mechanisms of DSB and GCR generation by palindromic sequences in yeast, Saccharomyces cerevisiae.

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Functional analysis of subtelomeric breakage motifs using yeast as a model organism

2011-05-24 , Khuzwayo, Sabelo Lethukuthula

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.

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