Access Type

Open Access Dissertation

Date of Award

January 2015

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Ashok S. Bhagwat

Abstract

Activation-induced deaminase (AID) is a sequence-selective DNA cytosine deaminase that introduces uracils in immunoglobulin genes. This DNA mutator is required for somatic hypermutation and class switch recombination- processes involved in the affinity maturation and diversification of antibodies. AID, however, can also lead to deleterious mutations and translocations promoting lymphomagenesis. The introduction of uracils throughout the genome of activated B cells and the ability of UNG2 glycosylase to excise these uracils is examined here. This interplay was also studied in cancerous B cells, with different results emerging in transformed cells versus healthy cells. Genomic uracil levels are found to remain at the same level in normal B cells stimulated to express AID. However the increase in uracils by 11- to 60- fold detected in stimulated B cells deficient in UNG, suggests that normal B cells do accumulate high levels of genomic uracils. However, these uracils are efficiently and effectively removed in UNG proficient cells, suggesting a balance or homeostasis between uracil creation and elimination. Interestingly, murine B cell cancer lines, human B cell cancer cell lines, and several human B cell patient tumors from several types of lymphomas overexpressing AID, were found to accumulate genomic uracils at levels comparable to those seen in activated UNG-/- B cells. These lymphoma/leukemia cells are not defective in uracil removal and express UNG2 gene at the same level in normal peripheral B cells or higher. In addition, they also have similar nuclear uracil excision activities suggesting uracils accumulate despite robust uracil excision capabilities. These results suggest that the homeostasis of uracil introduction and excision seen in normal stimulated B cells is disrupted in lymphomas overexpressing AID despite UNG2 expression and activity, resulting in the accumulation of high levels of genome wide uracils.

The majority of human B cell lymphomas do in fact derive from cells that have undergone the germinal center reaction and are associated with the expression of AID. The high levels of uracils that accumulate in the genomes of B cell lymphomas suggests other types of DNA damage may also be present in an AID-dependent manner as the cell attempts to remove and repair these uracils. Here, we see that human B cell lymphoma cell lines that contain high uracil loads also accumulate elevated levels of other types of genome wide DNA damage dependent on AID. These include abasic sites and single- and double-strand breaks. In addition, the accumulation of DNA lesions reduces cell viability. These lesions are due in part to the attempted repair of uracils by uracil DNA glycosylase.

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