RecQ-like proteins are a family of DNA helicases that are evolutionary conserved from prokaryotes to eukaryotes. A large amount of experimental evidence suggests these proteins have a major role in the maintenance of genome stability. In humans five RecQ like helicase have been identified (RecQL1, BLM, WRN, RecQL4, and RecQL5), three of which are associated with rare genetic disorders with sever chromosomal and developmental abnormalities, and an elevated predisposition to cancer. Among the disease associated RecQ-like helicases, BLM and WRN have been subject to extensive research, while our collective knowledge about the function of RecQL4 is still very limited. Similarly, little is known about the role of RecQL1 and RecQL5 in maintenance of genome integrity. In the past studies of Sgs1, the Saccharomyces cerevisiae homolog of RecQ, have been very informative regarding BLM function. Thus, here we sought to further investigate BLM, RecQL1, and RecQL4 by using yeast as a model. By constructing humanized yeast strains we evaluated the ability of these genes to complement defects observed in sgs1∆. In Chapter 2, our investigation led to the development of a novel chimeric system, which was able to complement some defects of the sgs1∆ strain. In Chapter 3, by taking advantage of this chimeric system, we evaluated 41 known BLM variants identified in the general human population. This study resulted in identification of six novel variants that completely impaired BLM function and three novel variants that partially impaired BLM function. In Chapter 4 we conducted multiple yeast 2-hybrid screens in search for novel protein-protein interaction for RecQL1 and RecQL4. We have identified two new putatively interacting partners for RecQL1 and three putatively interacting partners for RecQL4. In Chapter 5, functional characterization of RecQL1, BLM, RecQL4 and RecQL5 in yeast suggested genetic interaction between BLM and RecQL4 and RecQL5. Finally, in Chapter 6 a random mutagenesis screen of BLM has led to identification a mutation that impairs BLM function by disrupting the HRDC domain. This mutant suggests that the HRDC domain of BLM has an important role in proper functionality of this helicase.

Microsatellites (MSTs) are short tandem repeats of motifs, 1 — 6 nucleotide in length, and are considered mutational 'hot-spots' and show a greater degree of somatic variability and population polymorphisms than surrounding DNA sequences. MSTs provide for a unique computational alignment problem for many commonly used algorithms, and therefore required software tools to be developed to specifically address these issues. For this work we developed a novel approach to extract MSTs from next-gen sequencing data that can robustly detect signatures of MST mutation bias and somatic variation occurring in next-gen data including a high frequency of in-phase indels. Somatic variability, novel genomic polymorphisms that arise within a cell population not found in the progenitors, plays a critical role in cellular reprogramming leading to the development and progression of cancer. MST mutation rates are between 10 and 1000 time higher than that of surrounding DNA. MSTs are found ubiquitously throughout the genome including in nearly all transcribed regions and 10-20% of coding genomic regions. Currently the only established DNA repair defect that that has been directly linked to MST instability is replication coupled mismatch repair (MMR). An initial analysis of the utility of the software was conducted with DNA repair impaired cell lines. The results demonstrated the utility in identifying the consequences of DNA repair impairments on genomic stability. There were major objectives of the finding including 1) complimenting genomics of matched DNA samples with in-sample quantification of variation and 2) demonstrating that DNA repair proficient cells and those with different defects in DNA repair can have different somatic MST variability (SMV) profiles that may be potential markers for these defects. DNA repair disorders are debilitating conditions that result in physical and neurological abnormalities robbing the individual of a normal quality of life and life span. The various conditions that fall into this class are known as progeroid disorders and they provide a very important glimpse into the aging process on a genomic level. The conditions for four cohorts analyzed here were; Cockayne's syndrome, caused by the loss of the ERCC8 gene, also known as CSA; xeroderma pigmentosum, caused by the loss of the XPA or XPB genes; Werner syndrome, caused by the loss of the RecQL2 gene; and Rothmond-Thomson syndrome, caused by the loss of the RecQL4 gene. The goal of this project was to determine if impaired excision repair genes CSA or global XPA and B or excision repair supporting helicases BLM or RecQL4 leads to MST destabilization. Comparing cohorts from excision repair disorders with a co-sequenced normal cohort we found that CSA both RecQ helicases had an effect on the exome somatic variability of MSTs. On the other hand the effects of XPA/B were inconclusive. MST instability (MSI), defined as acquired/lost primary alleles in tumors for a small set of microsatellite loci, has been implicated and is a clinically relevant marker for colorectal cancer. Conversely, no clinically actionable genetic markers have been found for liver cancer, a cancer with a very high mortality rate. Here we explore the use SMV defined as the presence of minor alleles at MST loci, as a complementary measure of MSI as a genetic marker for colorectal and liver cancer by analyzing Illumina sequenced genomes from The Cancer Genome Atlas. Our data shows that SMV may distinguish a subpopulation of African American patients with colorectal cancer, ~33% of the population in this study. Further, for liver cancer, a higher rate of SMV may be indicative of earlier age of onset. In conclusion, the work presented here suggests that MSI should be expanded to include SMV, not only instability. / Ph. D.