Dissertation
Formation and Bypass of APOBEC-mediated DNA lesions
Doctor of Philosophy (PhD), Washington State University
01/2019
Handle:
https://hdl.handle.net/2376/17869
Abstract
AID/APOBEC family cytidine deaminases are established as endogenous DNA mutators that drive genomic instability in a wide array of human cancers. Many members of this family have a role in immunity through catalytically converting cytidines into uridines in single stranded DNA to increase immunoglobulin gene diversification or through hypermutation of viral intermediates. Recently DNA damage at trinucleotide motifs associated with APOBEC activity have come to spotlight as drivers of genetic instability in a wide array of human cancers, with mutations manifesting as C to T and C to G base substitutions in TCW sequences. We assessed how these single stranded DNA-targeting enzymes damage double stranded chromosomal DNA, and how the mutagenic consequences of these lesions are avoided by expressing human APOBEC3A and APOBEC3B in a yeast model system. In these yeasts, we analyzed the mutation frequency of the CAN1 forward reporter gene as well as the genome-wide mutational pattern associated with either replicational or transcriptional strand biases. The results presented here demonstrate that these enzymes predominately damage single stranded DNA intermediates formed on the lagging-strand template during DNA replication, with minimal discernable contribution from lesions on transcription intermediates. As these lesions occur in single stranded DNA at the replication fork, they are bypassed in an error-free manner by a Rad51-mediated template switching pathway following lesion conversion from a deoxyuridine to a fork-stalling abasic ahead of the replicative polymerase, rather than through an excision repair mechanism. This suggests that the observed mutagenic burden in cancer cells may be understated if cancers cells are reliant upon similar mechanisms to mitigate APOBEC-mediated damage. Furthermore, the results strongly indicate that APOBEC-mediated DNA damage contributes to destabilizing genomic integrity by generating mutations during DNA lagging-strand synthesis when bypass capacity is overwhelmed or incapacitated. A result of this could be the mutagenic activation of an oncogene or silencing of a tumor suppressor gene which could drive replication stress, exacerbating the mutagenic potential of APOBECs in a “perfect storm” of genomic instability. Understanding how APOBEC mutations are generated can better shed light on how cancers are generated, diversify, and gain drug resistance which leads to tumor recurrence.
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Details
- Title
- Formation and Bypass of APOBEC-mediated DNA lesions
- Creators
- James Isamu Hoopes
- Contributors
- Steven A. Roberts (Advisor)Terry Hassold (Committee Member)Chengtao Her (Committee Member)John Hinz (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- School of Molecular Biosciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 128
- Identifiers
- 99900581615101842
- Language
- English
- Resource Type
- Dissertation