Introducing tumor-derived cells into normal mammary stem cell niches at a sufficiently high ratio of normal to tumorous cells causes those tumor cells to undergo a change to normal mammary phenotype and yield normal mammary progeny. This phenomenon has been termed cancer cell redirection. We have developed an in vitro model that mimics in vivo redirection of cancer cells by the normal mammary microenvironment. Using the RNA profiling data from this cellular model, we examined high-level characteristics of the normal, redirected, and tumor transcriptomes and found the global expression profiles clearly distinguish the three expression states. To identify potential redirection biomarkers that cause the redirected state to shift toward the normal expression pattern, we used mutual information relationships between normal, redirected, and tumor cell groups. Mutual information relationship analysis reduced a dataset of over 35,000 gene expression measurements spread over 13,000 curated gene sets to a set of 20 significant molecular signatures totaling 906 unique loci. Several of these molecular signatures are hallmark drivers of the tumor state. Using differential expression as a guide, we further refined the gene set to 120 core redirection biomarker genes. The expression levels of these core biomarkers are sufficient to make the normal and redirected gene expression states indistinguishable from each other but radically different from the tumor state.

Uracil DNA glycosylase (UDG) is an enzyme to initiate base excision repair (BER) of DNA by removing the damaged base from the strand and forming an apurinic/apyrimidinic site (AP site). There are at least six families of enzymes found in UDG superfamily. All the UDGs contain three motifs in their catalytic centers, and the sequences of the motifs are conserved, guaranteeing the glycosylase activity. UDGX was a newly discovered protein of the UDG superfamily from Mycobacterium smegmatis, which was confirmed to be closer to the Family 4 UDGs (UDGa) than the other members by studying its structure and sequence. However, different with the Family 4 UDGs, an obvious loop was inserted in the motif 3 of UDGX. In addition, UDGX was the first reported UDG which can covalently crosslink to the AP site formed by base excision via its His109 on the inserted loop. Several mutants were generated by substituting the key residues in the motif 1 and motif 3 of the MsmUDGX. Uracil DNA glycosylase activity assays as well as uracil DNA crosslinking activity assays were performed with these mutants. It was observed that the E52A and D56A mutants retained both the uracil DNA glycosylase and DNA crosslinking activities, however the H109A, H109N and H109Q only retained the uracil DNA glycosylase activity but lost the DNA crosslinking activity due to the substitution of the His109. Moreover, the loss of both activities of the D59A and R107A mutants were observed. Followed by the activity assays, kinetic analyses were performed on the mutants and the UDGX wild type. The k2/Km of the wild type MsmUDGX on the G/U substrate was determined to be 1.8 ± 0.2 × 104 s‐1M‐1, which was 10‐fold lower than the k2/Km of the Family 4 UDG (TthUDGa) reported. Compared to the wild type, E52A, H109A, H109N and H109Q mutants exhibited a significant decrease of the catalytic reaction rate, suggesting that not only the key residue Glu52 in the motif 1 but His109 may also play an important role in the uracil excision process. The Asp56 and Asp59 in the motif 1 were confirmed to form salt bridges with the Arg107 on the inserted loop in the motif 3, which were hypothesized to stabilize the loop during the interaction between the enzyme and the DNA substrates. Consistent with the hypothesis, the mutations on Asp59 and Arg107 caused the loss of both activities, but the D56A mutant retained both activities, and the kinetic analysis indicated only slight decrease of the reaction rate, which may suggest the Asp56 was not the key residue to stabilize the loop in the salt bridges. In conclusion, the kinetic analysis provides a quantitative assessment on the roles of key catalytic residues on uracil excision activity and DNA‐crosslinking activity.

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Uracil‐DNA glycosylase (UDG) initiates base excision repair (BER) by removing damaged or modified nucleobases during DNA repair or mammalian demethylation. The UDG superfamily consists of at least six families with a variety of catalytic specificities and functions. Deinococcus radiodurans, an extreme radiation resistant bacterium, contains multiple members of UDG enzymes within its genome. The present study reveals that the putative protein, DR0022, is a uracil‐DNA glycosylase that requires acidic conditions for its glycosylase activity, which is the first case of such an enzyme within the UDG superfamily. The key residues in the catalytic motifs are investigated by biochemical, enzyme kinetics, and de novo structural prediction, as well as molecular modeling analyses. The structural and catalytic roles of several distinct residues are discussed in light of predicted and modeled DR0022 glycosylase structures. The spontaneous mutation rate analysis performed in a dr0022 deficient D. radiodurans strain indicated that the dr0022 gene plays a role in mutation prevention. Furthermore, survival rate analysis in a dr0022 deficient D. radiodurans strain demonstrated its role in stress resistance, including γ‐irradiation. Additionally, the novel acid UDG activity in relationship to its in vivo roles is discussed. This work underscores the functional diversity in the UDG superfamily.

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