Microsatellites as inducers of genome instability: Studies into the mechanisms of replication and repair of repetitive DNA

Open Access
- Author:
- Shah, Sandeep
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 08, 2009
- Committee Members:
- Kristin Ann Eckert, Dissertation Advisor/Co-Advisor
Kristin Ann Eckert, Committee Chair/Co-Chair
Gavin Peter Robertson, Committee Member
Sergei A Grigoryev, Committee Member
Leslie Joan Parent, Committee Member
Lisa M Shantz, Committee Member - Keywords:
- primer extension
polymerase
WRN
replication
FRA16D
PMS2
Mismatch Repair
Fragile sites
HNPCC
mutation bias - Abstract:
- Genomic instability is a hallmark of tumor initiation and progression. A tumor genome can contain both gross chromosomal alterations as well as multiple submicroscopic nucleotide changes. Indeed, it is well established that multiple mutations are necessary for a cell to become tumorigenic. In this dissertation, I have studied two major cellular processes that, under normal circumstances, largely suppress the initial events that favor these alterations. These two processes are DNA replication, which duplicates the genome in order to fully distribute all genetic information to two daughter cells, and DNA mismatch repair, which corrects a subset of errors that occur during replication. DNA replication is an extremely accurate process where, on average, mutations occur once every 109-1010 base pairs during cell division. The central proteins for replication are DNA polymerases which travel along the template DNA while extending nascent DNA by nucleotide addition. While the vast majority of DNA is in the right-handed double helix form, many other conformations can exist within particular sequence arrangements. These non-canonical structures can impede the polymerase and stall replication until secondary mechanisms restart the process. Specific chromosomal areas have been found that are especially prone to replicative stress. These areas, termed common fragile sites (CFS), exhibit chromosomal gaps and breaks during replicative challenge and may represent a mechanism for initiating genomic instability. Chromosomal breaks are a prerequisite for alterations such as translocations, large deletions, and amplifications found in advanced tumor cells. CFS are also among the last regions to be replicated during culture of healthy untreated cells. Studies have determined that the majority of CFS are AT rich, a property believed to cause increased torsional stress and DNA secondary structure formation. This finding has lead to the hypothesis that CFS expression (breakage) may be due, in part, to stalling of the polymerase complex. Although DNA replication is a very accurate process on average, certain regions – e.g., microsatellites – have a very high error rate. Microsatellites are short, repetitive elements in the DNA consisting of 1-6 bases per repeat unit. The repetitive nature of these sequences can provoke length alterations due to strand slippage events during replication. The human DNA mismatch repair (MMR) system, a specialized repair mechanism, plays a major role in post-replicative repair of DNA polymerization errors. Loss of this pathway results in hereditary cancers characterized by microsatellite instability. In this dissertation, I first describe our studies of hPMS2, a component of MMR, and observe its role in error correction of mono-, di-, and tetranucleotide repeat motifs. Alterations at mono- and dinucleotide microsatellites have been extensively used to diagnose hereditary nonpolyposis colorectal cancer, while tetranucleotide microsatellites have been utilized for the detection of another, mechanistically different type of instability termed EMAST. While microsatellite alterations may be a general consequence of genomic instability post tumor initiation, they may also directly promote tumor progression. Using a shuttle vector assay in human cells, I determined that the mutation rate of vectors containing [G/C]10 or [GT/CA]10 alleles was elevated 20-fold to 40-fold in hPMS2-deficient cells, relative to an hPMS2-expressing cell line. In contrast, a 6-fold or 12-fold relative mutation rate increase was observed in [TTTC/AAAG]9 and [TTCC/AAGG]9-containing vectors, respectively. These data demonstrate that the corrective ability of the MMR complex containing PMS2 is more protective of tetranucleotide expansions than deletions. hPMS2 also displays a sequence bias, wherein [TTCC/AAGG] sequences are stabilized to a greater extent than [TTTC/AAAG]. These results have the potential to add greater accuracy to microsatellite panels used for cancer diagnosis by the addition of highly mutable alleles. In chapter 3, I focus on the mechanism of CFS expression by determining sequence specific effects on polymerase progression. I have chosen to study DNA polymerase delta (pol δ) as it is suggested to be primarily involved in lagging strand synthesis, and secondary structure formation is more likely on the lagging strand, since the DNA is more often single stranded. I have also extended these findings by studying the DNA polymerase alpha-primase complex due to its emerging role in synchronizing leading/lagging strand synthesis as well as checkpoint signaling upon fork arrest. Using in vitro primer extension assays, I found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and between complementary strands. Both polymerases were significantly inhibited in regions containing hairpins and microsatellites, [AT/TA]24 and [A/T]19-28, compared with a control region with minimal secondary structure. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. These data support a model whereby CFS expression during cellular stress is due to a combination of factors, including the density of specific DNA secondary structures within a genomic region and asymmetric rates of strand synthesis. Finally, I describe the ability of the Werner helicase to aid polymerase progression and prevent polymerase stalling within CFS. Since WRN contains multiple enzymatic activities and has been shown to be necessary for preventing chromosomal breaks within CFS, I sought to determine whether WRN aids CFS replication simply by unwinding DNA secondary structure or through an alternate pathway. Using defined WRN fragments, I conclude that WRN can enhance pol δ processivity with and without functional helicase activity possibly as a result of direct binding and stimulation of the polymerase/PCNA complex. Overall, the work described in this dissertation offers novel insight into the role of microsatellites as predeterminants of cancer. The significance of microsatellites within inherently unstable genomic areas was elucidated and a potential mechanism that assists replication within these areas was discovered. The use of this information has vast therapeutic potential, including the induction of instability in cancer cells or modification of current gene therapy approaches to reduce negative side effects. Finally, studies into the mechanisms that govern the maintenance and repair of microsatellites showed important repair biases that can be exploited for genetic counseling and cancer diagnosis.