A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.

Recombination and mutagenesis are elevated by active transcription. The correlation between transcription and genome instability is largely explained by the topological and structural changes in DNA and the associated physical obstacles generated by the transcription machinery. However, such explanation does not directly account for the unique types of mutations originating from the non-canonical residues, uracil or ribonucleotide, which are also elevated at highly transcribed regions. Based on the previous findings that abasic (AP) lesions derived from the uracil residues incorporated into DNA in place of thymine constitute a major component of the transcription-associated mutations in yeast, we formed the hypothesis that DNA synthesis ensuing from the repair of the transcription-induced DNA damage provide the opportunity for uracil-incorporation. In support of this hypothesis, we show here the positive correlation between the level of transcription and the density of uracil residues in the yeast genome indirectly through the mutations generated by the glycosylase that excise undamaged cytosine as well as uracil. The higher uracil-density at actively transcribed regions is confirmed by the long-amplicon PCR analysis. We also show that the uracil-associated mutations at a highly transcribed region are elevated by the induced DNA damage and reduced by the overexpression of a dUTP-catalyzing enzyme Dut1 in G1- or G2-phases of the cell cycle. Overall, our results show that the DNA composition can be modified to include higher uracil-content through the non-replicative, repair-associated DNA synthesis.

Loss of KDM6A promotes pancreatic cancer progression and metastasis and delays pancreatic tissue recovery from pancreatitis. Mechanistically, a noncanonical p38-dependent activin A pathway likely mediates KDM6A function.BACKGROUND & AIMS: Inactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A in PDAC development. METHODS:We performed a pancreatic tissue microarray analysis of KDM6A Q8

Most B cell cancers overexpress the enzyme activation-induced deaminase at high levels and this enzyme converts cytosines in DNA to uracil. The constitutive expression of this enzyme in these cells greatly increases the uracil content of their genomes. We show here that these genomes also contain high levels of abasic sites presumably created during the repair of uracils through base-excision repair. We further show that three alkoxyamines with an alkyne functional group covalently link to abasic sites in DNA and kill immortalized cell lines created from B cell lymphomas, but not other cancers. They also do not kill normal B cells. Treatment of cancer cells with one of these chemicals causes strand breaks, and the sensitivity of the cells to this chemical depends on the ability of the cells to go through the S phase. However, other alkoxyamines that also link to abasic sites- but lack the alkyne functionality- do not kill cells from B cell lymphomas. This shows that the ability of alkoxyamines to covalently link to abasic sites is insufficient for their cytotoxicity and that the alkyne functionality may play a role in it. These chemicals violate the commonly accepted bioorthogonality of alkynes and are attractive prototypes for anti-B cell cancer agents.