Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W ‫؍‬ A/T, R ‫؍‬ A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd) and raltitrixed (RTX), are anticancer agents that target thymidylate synthase (TS) thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG) leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated the how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1 and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP/dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth is in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U/A base pairs derived from dUTP incorporation, an isogenic ung−/− DT40 cell line showed little change its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A, G or present in ssDNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, nature of the resulting DNA lesion, and the DNA repair response are discussed.The antimetabolites 5-fluorouracil (5-FU) 1 , 5-flurodeoxyuridine (5-dUrd) and raltitrexed (RTX) are widely used for the treatment of colorectal, breast, and head and neck cancers (1, 2,3). Fluoropyrimidines are metabolized much like uracil and deoxyuridine and can be enzymatically converted to the active metabolite 5-FdUMP (Fig. 1). A binary complex between 5-FdUMP and 5,10-methylenetetrahydrofolate irreversibly inhibits thymidylate synthase (TS), blocking de novo production of dTMP and also resulting in accumulation of dUMP. The † This work was supported by grant GM056834 from the National Institutes of Health to J.T.S. and a Ruth L. Kirschstein National Research Service Award (F31 GM083623) to J. B. P. * To whom correspondence should be addressed. Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185. Telephone: 410-502-2758. jstivers@jh...