In partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Biology
In the
School of Biological Sciences
Alex Costa
Will defend his dissertation
MECHANISMS OF PALINDROME-MEDIATED
CHROMOSOMAL INSTABILITY
Thursday, June 20th, 2024
10:00 AM
Kendeda Building, Room 230
Thesis Advisor:
Kirill Lobachev, Ph.D.
School of Biological Sciences
Georgia Institute of Technology
Committee Members:
Yury Chernoff, Ph.D.
School of Biological Sciences
Georgia Institute of Technology
Francesca Storici, Ph.D.
School of Biological Sciences
Georgia Institute of Technology
Matthew Torres, Ph.D.
School of Biological Sciences
Georgia Institute of Technology
Amit Reddi, Ph.D.
School of Chemistry & Biochemistry
Georgia Institute of Technology
ABSTRACT: Palindromes are highly unstable DNA sequences that induce chromosomal rearrangements and underlie cancer and hereditary diseases such as Emanuel Syndrome. Despite being a significant source of genomic instability, the mechanisms governing palindrome fragility in eukaryotic cells are not fully defined. I discovered that palindromic sequences with nonpalindromic spacers of 8 bp or less form stable hairpins which can block replication, and these hairpins are cleaved by the Mre11/Rad50/Xrs2/Sae2 complex. In contrast, palindromic sequences with spacers longer than 8 bp are not cleaved by MRX/Sae2 and do not block replication fork progression. I show that these effects are mediated by single stranded DNA binding protein RPA, which is likely facilitating hairpin unwinding. I also describe a novel pathway of palindrome-mediated chromosomal instability involving Mph1, Rad51, Rad54, Smc5, and Smc6. Deletion or mutation of any of these genes results in a ~3-fold reduction in palindrome-mediated breaks. I propose that Mph1, Rad51, and Rad54 mediate fork remodelling at stalled replication forks, generating cruciform structures and that the Smc5/6 holo-complex stabilizes these structures and facilitates cleavage by Mus81/Mms4 and other resolvases. Last, I described the Lobachev Lab’s progress in creating a novel, antibiotic resistance-based gross chromosomal (GCR) and gene amplification assay in human cells. The human GCR assay will enable foundational work done in model organisms regarding palindrome instability to be extended into the realm of clinical application and enable testing of human specific factors. Defining the pathways and mechanisms that govern palindrome instability is essential for understanding how pathogenic genomic rearrangements occur.