Research in Bhagwat Lab
Functional Genomics of Uracils in DNA
AID/APOBEC enzymes1 in mammals are DNA-cytosine deaminases that play a key role in innate and adaptive immunity against infections by bacteria, fungi and viruses. Uracils created by these enzymes have three possible fates: (1) They may be repaired through base-excision repair (BER) to restore C:G pairs and prevent mutations; (2) They may be replicated causing C:G to T:A mutations; and (3) The uracil-DNA glycosylase (UNG) may excise the uracils and the resulting abasic sites may be bypassed by translesion synthesis (TLS) polymerases, creating all six possible base substitution mutations. Consequently, a complete molecular description of what AID/APOBECs do to the genome is not possible without directly studying the fate of the pro-mutagenic lesion created by them, uracil. To accomplish this, we are developing a variety of tools to quantify and map uracils in genomes. Our goal is to study the dynamics of uracil distribution in mammalian genomes (the “uracilome”) and correlate with molecular and biological consequences such as mutations, strand breaks, chromosome translocations and carcinogenesis.
Quantification of uracils: We quantify uracils using a chemical that reacts with abasic sites in DNA and a fluorescent reporter that is activated through Cu-free click chemistry. The principle of the assay is shown below.
The fluorescence signal from a genomic DNA sample is converted to number of uracils/106 bp using a standard that is validated using LC/MS/MS. This procedure has been used to quantify abasic sites and uracils in a number of human cancer cell lines2,3 and is being used to determine the DNA damage caused by AID/APOBEC enzymes in normal and cancer cells.
Mass chromatogram of isotopically labelled uracil and unlabeled uracil
Comparison of abasic (AP) sites in different normal and cancer cell lines
Visualization of genomic uracils: An unusual protein from Mycobacterium smegmatis, UdgX, links covalently to uracils in DNA. The protein does not bind normal DNA bases and the resulting complexes are stable under strong denaturing conditions. We are using this fluorescently tagged UdgX to directly map uracils in the mammalian genomes.
A. Purification of UdgX protein
B. Covalent complex between UdgX and uracil-containing DNA
Visualization of UdgX expressed in HeLa cells
Mapping of uracils in genomic DNA: We have now extended the technology developed to quantify uracils in DNA to mapping uracils at genomic level. The novel procedure involves pull-down of uracilcontaining DNA fragments and mapping them to the whole genome using bioinformatic analysis. Using this technology, we have mapped uracils created by the human APOBEC3A in the E. coli genome. We are now extending this “proof-of-principle” result to mapping uracils created by AID in a mammalian genome.
Distribution of uracils created by APOBEC3A in the E. coli genome
Positions of uracil peaks in protein-coding genes
Specific killing of B cell cancers by alkoxyamines: We have found that when some alkoxyamines link covalently to abasic sites created by AID/APOBEC enzymes in human cancer genomes, the cells die. This phenomenon requires the alkoxyamine moiety as well as an alkene or an alkyne in the molecule. These alkoxyamines kill B cell-derived cancer cells, but do not kill other types of cancers. We are now developing these chemicals further to increase their effectiveness and to determine their mechanism of action.
A. Relative killing of a Burkitt lymphoma cell line by different alkoxyamines
B. Lack of killing of non-hematological cancers by AA3