Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Ashok S. Bhagwat

Abstract

The AID/APOBEC family of proteins in higher vertebrates converts cytosines in DNA or RNA into uracil. These proteins have essential roles in either innate immunity or adaptive immunity. Recently, AID has also been implicated in DNA demethylation in the context of early embryogenesis in mammals. This is partly based on the reported ability of AID to deaminate 5-methyl cytosine to thymine (5mC to T). I reexamined this proposed new role of AID (5mC deamination) with two members of the APOBEC family in a novel Escherichia coli based genetic system. My results confirmed that while all three enzymes are strong cytosine deaminases, only APOBEC3A (A3A) is an efficient deaminator of 5mC. The partially purified A3A converted 5mC to T in vitro as well. This is the first report of efficient deamination of 5mC by any enzyme from any organism. Although AID did not deaminate 5mC efficiently, when the DNA binding region of AID was replaced with the corresponding segment from A3A, the resulting hybrid protein deaminated 5mC efficiently. Together these results suggest that human AID deaminates 5mCs very weakly. Consequently, AID is unlikely to promote genome-wide DNA demethylation unless its ability to deaminate 5mC is enhanced through covalent modifications or accessory factors.

APOBECs are DNA or RNA binding proteins with substrate preferences. APOBEC3G targets the last cytosine (C) in a run of Cs (usually three Cs) whereas AID prefers to act on WRC (where W is A or T and R is purine) in DNA. Guided by the solution structures of the APOBEC3G carboxy terminal domain (A3G-CTD), two putative DNA binding regions (R1 and R2) were recognized in the A3G-CTD structure. These potential DNA binding regions of A3G-CTD were swapped with the corresponding regions in AID. The biochemistry of the hybrid proteins A3G-AIDR1 (A3G protein containing AID DNA binding region 1), A3G-AIDR2 and A3G-AIDR1R2 were studied using an in vitro deamination assay. The data revealed that regions 1 and 2 of AID are not sufficient to confer its sequence-specificity upon A3G. Instead, replacement of Region 2 of A3G with corresponding region from AID broadens the specificity of A3G to act on WRC in addition to CCC.

Download

Share

COinS