I. Design, synthesis, and characterization of peptides for thermodynamic binding studies of PDZ protein interaction domains II. DNA cytosine deaminases and the development of assays to study uracil in the genome.

摘要

The presence of uracil in DNA is largely a result of cytosine deamination or misincorporation of dUMP in place of TTP during replication. This results in the presence of either mutagenic U:G mispairs or the comparatively benign U:A pair in genomic DNA, respectively. The dire implication of having elevated levels of uracil in the genome is evident in the fact that there are at least four human DNA glycosylases tasked with removing it. These are: UNG, SMUG1, TDG, and MBD4. The excision of uracil itself is not without its own danger, as this results in the generation of abasic sites which are also potentially mutagenic. Under normal conditions, however, cells are readily able to excise the uracil base and correctly restore cytosine in its place. This is the foremost reason why steady-state uracil levels in genomic DNA are low, making detection difficult.;There is a singular exception to the otherwise negative effects of having uracil in the genome. During antibody maturation, activation-induced deaminase (AID) purposely deaminates cytosines in the immunoglobulin (Ig) genes. This results in two processes required for antibody maturation, called somatic hypermutation (SHM) and class switch recombination (CSR). Deficiencies in AID lead to defects in the immune system and greater susceptibility to infections. On the other hand, aberrant expression of AID has been implicated in genomic instability and cancer. APOBEC3G (A3G) is a cytosine deaminase that like AID, belongs to the APOBEC super family. Rather than acting on cellular DNA however, A3G deaminates cytosines in the negative strand of retroviral DNA such as HIV, thus interfering with the production of viable virions. Aberrant expression of this enzyme may have the same ominous repercussions as with AID.;Several studies clearly implicate uracil as a necessary intermediate resulting from AID- and A3G-induced cytosine deamination in antibody maturation and antiviral action respectively, however thus far there has not been any direct evidence to prove this assumption and certainly none pinpointing the location of uracils to specific regions of the genome. This research describes two assays developed specifically for these purposes. The uracil dot blot assay and the uracil southern blot assay are powerful tools that can be used for studying antibody maturation, retroviral restriction, and APOBEC associated mutation and carcinogenesis.