Repetitive DNA sequence elements and DNA polymerases modulate instability in the human genome

The goals of this thesis are to: 1) investigate the mutational behaviors of microsatellite repeats, in order to gain a better understanding of the mechanisms by which microsatellite instability is driven in the human genome; and 2) investigate the types of repetitive sequence elements and replication proteins modulating chromosomal instability at regions of the human genome known as common fragile sites (CFS).;In Chapter 2, direct comparison of computationally derived human polymorphism incidence rates and experimentally determined polymerase slippage rates within tandem repeat sequences was carried out to determine the mechanisms contributing to the mutability of mononucleotide and dinucleotide microsatellites in the human genome. Experimentally determined polymerase error frequencies (Pol EF), representing polymerase insertion and deletion errors within tandem repeat tracts, were plotted as a function of repeat length alongside a curve representing log-scale polymorphism incidence versus repeat length for mono- and dinucleotide tandem repeats. Interestingly, these curves displayed significant overlap for mononucleotide tandem repeats, suggesting that mononucleotide tandem repeat mutability might be driven largely by polymerase slippage-based errors. In contrast, for dinucleotide tandem repeats, significant differences were observed for the dinucleotide tandem repeat polymorphism incidence and Pol EF curves, suggesting that cellular mechanisms other than polymerase slippage contribute to dinucleotide tandem repeat mutability. Furthermore, I present experimentally determined data demonstrating that polymerase slippage errors within tandem repeats depend on the identity of the DNA polymerase. .;In Chapter 3, I test the hypothesis that enrichment of specific repetitive sequence elements at CFS regions contributes to fragility through replication perturbation by directly impeding the replicative DNA polymerases. To test this hypothesis, the efficiency of in vitro DNA synthesis by the four subunit, lagging strand DNA polymerase delta (Pol delta) was quantified using templates corresponding to regions within FRA16D and FRA3B harboring specific repetitive elements, in the presence and absence of replication accessory proteins. Analysis of DNA synthesis progression by human Pol delta demonstrated significant synthesis perturbation both at [A]n and [TA]n repeats in a length-dependent manner, and at short (<40 base pairs) quasi-palindrome (inverted repeat) sequences. Using DNA trap experiments, I show that Pol delta pauses within CFS sequences are sites of enzyme dissociation, and dissociation occurs in the presence of RFC-loaded PCNA. Using a 3'→5' exonuclease defective Pol delta mutant, I show that pausing is decreased relative to the wild type Pol delta. This suggests that Pol delta pausing at predicted CFS hairpin structures might involve 3'→5' exonuclease idling.;In Chapter 4, I test the hypothesis that Pols kappa and eta are important for maintaining efficient replication of CFS sequences through synthesis of repetitive DNA sequences. To test this hypothesis, DNA synthesis efficiency through CFS templates containing repetitive DNA sequence elements was determined for the specialized Pols kappa and eta using a primer extension assay, and compared to the replicative Pol delta. Pol eta displayed significantly decreased levels of pausing, particularly at predicted hairpin structures, which correlated with greater rates of synthesis through CFS templates than Pol delta. The specialized Pol kappa showed the least pausing at mononucleotide microsatellites, and this also corresponded with significantly greater synthesis rates than Pol delta. Together, our findings of DNA synthesis dynamics within CFS sequences support a model whereby sequence elements with non-B potential impede the replicative Pol, and specialized Pol functions are required to prevent breakage.;In Chapter 5 I present the development of an ex vivo replication assay in human cells, which will be of great value for future studies of repetitive DNA sequences. In this chapter, I also investigate the question: what is the minimal repetitive sequence element(s) sufficient to cause replication stalling in cells? My findings suggest that although single CFS repetitive sequence elements are sufficient to contribute to pausing by the replicative Pols delta and epsilon in vitro, cellular replication stalling might require clustering of additional repetitive elements. Alternative models exploring the mechanisms by which repetitive sequence elements contribute to CFS instability are developed in Chapters 5 and 6. (Abstract shortened by UMI.).