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Access Type
WSU Access
Date of Award
January 2023
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Ashok Bhagwat
Abstract
The human APOBEC3 family of enzymes exhibit diverse biological functions, substrates and target sequence specificities, and sub-cellular localizations. Among these enzymes, APOBEC3A and APOBEC3B are found to be the main sources of APOBEC-signature mutations in many different cancers. To understand individual roles of these enzymes in mutagenesis and to evaluate their preferential target sites in the genome, the uracils created by APOBEC3A(A3A) in the genome were recently mapped using the uracil pulldown and sequencing (UPD-Seq) technique. Here I report the mapping of targets of a catalytically active, APOBEC3B carboxy-terminal domain (A3B-CTD) and full length A3B (A3B FL), in Escherichia coli. There were many similarities and differences between the targets of A3A and A3B. Like A3A, the uracils were found in distinct peaks that preferentially overlapped transcription start sites of tRNA genes. A majority of the target genes of A3B-CTD were also targets of A3A, and-like A3A, A3B preferentially targeted the lagging-strand template, demonstrating a replication strand bias. These similarities probably reflect the strong sequence and structural similarities between the two enzymes. Unexpectedly, our analysis found that like A3A, A3B also preferentially targets hairpin structures. This is a surprise because the full-length A3B has been shown previously not to strongly prefer hairpin loop structures. Data from my research analyses shows A3B’s preference for hairpin loop length, cytosine position in the loop, and the effect of stem strength on uracilation. Additionally, it is shown that although APOBEC3B preferentially targets TpC sequences in hairpins, it does not preferably target VpC sequences with that high preference as A3A does. I have also confirmed optimal hairpin loop preferences of purified A3B-CTD protein. Moreover, it is determined that binding plays a partial role in selectivity of optimal DNA substrates. Future studies will aim at studying APOBEC3A and APOBEC3B over-expressed in mammalian cells to determine the target preferences of these enzymes in actual environment. This will help understand how unique and similar these enzymes are in their targeting within actual environment. Moreover, kinetics studies can be performed for optimal vs poor substrates to further understand substrate selectivity for these enzymes.
Recommended Citation
Butt, Yasha Nazir, "Substrate Selection For Deamination By Human Apobec3a And Apobec3b Enzymes" (2023). Wayne State University Dissertations. 3919.
https://digitalcommons.wayne.edu/oa_dissertations/3919