Title:
Understanding the mechanisms and consequences of DSB induced by palindromic DNA sequences
Understanding the mechanisms and consequences of DSB induced by palindromic DNA sequences
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Sheng, Ziwei
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Abstract
Palindromic DNA sequences, capable of forming hairpin or cruciform structures, are often a potent sources of genome instability by inducing double strand breakage (DSB). Studies on palindrome-induced instability have proposed several mechanisms in which different nucleases, responsible for inducing DSBs at the repeats, are involved. The lack of consensus from the conclusions drawn from these studies can be attributed to a variety of factors, such as using different model organisms and repeats with various features. Here, I systematically demonstrate that there are three pathways governing the instability at palindromic repeats. Sae2-MRX nuclease plays a dual role of recognizing and attacking structures forming at perfect palindromic repeats during replication and processing and opening hairpin-capped ends for resection. However, Sae2-MRX does not affect quasi-palindrome fragility. Structure-specific endonuclease Mus81-Mms4 is responsible for inducing DSB at transcription-active palindromes in G2 stage of cell cycle and results in hairpin-capped breaks. The third pathway, which is Sae2-MRX- and Mus81-Mms4-independent, also happens in G2 stage and is under control of cell cycle kinase Cdc28 and LSm proteins that are involved in mRNA degradation. Moreover, I demonstrated that palindromes induce mutagenesis in the surrounding regions and Sae2-MRX-induced DSBs are channeled into break-induced replication (BIR). The synthesis intermediate with BIR characteristics, ‘bubble’-shape, was detected at both sides of breakage and at distances over 100kb away, indicating that the broken ends can anneal to the complementary strand on both Watson and Crick strands using repeat’s homology and initiate bidirectional BIR. The progression of BIR is stable and bypasses replication origins and the centromere. We also analyzed the players involve in palindrome-induced BIR and found that Pif1 helicase and Rad52 recombinase are required during the process. This study provides a systematic and mechanistic insight into the palindrome-induced genome instability. In another study, I participated in collaboration study with Dr. Anna Malkova’s laboratory in University of Iowa, we demonstrated that Srs2 helicase is involved in resolving toxic recombination intermediates during HO endonuclease-induced BIR. I showed that complex DNA structures other than ‘bubble’-like BIR structures are accumulated in srs2Δ mutants by two-dimensional gel electrophoresis. In srs2Δ mus81Δ double mutant, we can observe structures resemble ‘X’-shape recombination intermediates, indicating that Mus81/Mms4 is involved in resolving the recombination intermediates in the absence of Srs2. This study proposed the important role of the Srs2 helicase in break-induced repair.
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Date Issued
2017-04-07
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Dissertation