In budding yeast the cullin Rtt101 promotes replication fork progression through natural pause sites and areas of DNA damage, but its relevant subunits and molecular mechanism remain poorly understood. Here, we show that in budding yeast Mms1 and Mms22 are functional subunits of an Rtt101‐based ubiquitin ligase that associates with the conjugating‐enzyme Cdc34. Replication forks in mms1Δ, mms22Δ and rtt101Δ cells are sensitive to collisions with drug‐induced DNA lesions, but not to transient pausing induced by nucleotide depletion. Interaction studies and sequence analysis have shown that Mms1 resembles human DDB1, suggesting that Rtt101^Mms1 is the budding yeast counterpart of the mammalian CUL4^DDB1 ubiquitin ligase family. Rtt101 interacts in an Mms1‐dependent manner with the putative substrate‐specific adaptors Mms22 and Crt10, the latter being a regulator of expression of ribonucleotide reductase. Taken together, our data suggest that the Rtt101^Mms1 ubiquitin ligase complex might be required to reorganize replication forks that encounter DNA lesions.

Hat1 is the catalytic subunit of the only type B histone acetyltransferase known (HAT-B). The enzyme specifically acetylates lysine 12, and to a lesser extent lysine 5, of free, non-chromatin-bound histone H4. The complex is usually isolated with cytosolic fractions and is thought to be involved in chromatin assembly. The Saccharomyces cerevisiae HAT-B complex also contains Hat2, a protein stimulating Hat1 catalytic activity. We have now identified by two-hybrid experiments Hif1 as both a Hat1- and a histone H4-interacting protein. These interactions were dependent on HAT2, indicating a mediating role for Hat2. Biochemical fractionation and co-immunoprecipitation assays demonstrated that Hif1 is a component of a yeast heterotrimeric HAT-B complex, in which Hat2 bridges Hat1 and Hif1 proteins. In contrast to Hat2, this novel subunit does not appear to regulate Hat1 enzymatic activity. Nevertheless, similarly to Hat1, Hif1 influences telomeric silencing. In a localization analysis by immunofluorescence microscopy on yeast strains expressing tagged versions of Hat1, Hat2, and Hif1, we have found that all three HAT-B proteins are mainly localized in the nucleus. Thus, we propose that the distinction between A- and B-type enzymes should henceforth be based on their capacity to acetylate histones bound to nucleosomes and not on their location within the cell. Finally, by Western blotting assays, we have not detected differences in the in vivo acetylation of H4 lysine 12 (acK12H4) between wild-type and hat1Δ, hat2Δ, or hif1Δ mutant strains, suggesting that the level of HAT-B-dependent acK12H4 may be very low under normal growth conditions.

Class I _A phosphoinositide 3-kinase (PI3K) are enzymes comprised of a p85 regulatory and a p110 catalytic subunit that induce formation of 3-polyphosphoinositides, which activate numerous downstream targets. PI3K controls cell division. Of the 2 ubiquitous PI3K isoforms, α has selective action in cell growth and cell cycle entry, but no specific function in cell division has been described for β. We report here a unique function for PI3Kβ in the control of DNA replication. PI3Kβ regulated DNA replication through kinase-dependent and kinase-independent mechanisms. PI3Kβ was found in the nucleus, where it associated PKB. Modulation of PI3Kβ activity altered the DNA replication rate by controlling proliferating cell nuclear antigen (PCNA) binding to chromatin and to DNA polymerase δ. PI3Kβ exerted this action by regulating the nuclear activation of PKB in S phase, and in turn phosphorylation of PCNA negative regulator p21 ^Cip . Also, p110β associated with PCNA and controlled PCNA loading onto chromatin in a kinase-independent manner. These results show a selective function of PI3Kβ in the control of DNA replication.

Our main interest is the characterization of compounds to support the development of alternatives to currently marketed drugs that are losing effectiveness due to the development of resistance. Schiff bases are promising biologically interesting compounds having a wide range of pharmaceutical properties, including anti-inflammatory, antipyretic, and antimicrobial activities, among others. In this work, we have synthesized 12 Schiff base derivatives of 4-aminoantipyrine. In vitro antimicrobial, antioxidant, and cytotoxicity properties are analyzed, as well as in silico predictive adsorption, distribution, metabolism, and excretion (ADME) and bioactivity scores. Results identify two potential Schiff bases: one effective against E. faecalis and the other with antioxidant activity. Both have reasonable ADME scores and provides a scaffold for developing more effective compounds in the future. Initial studies are usually limited to laboratory in vitro approaches, and following these initial studies, much research is needed before a drug can reach the clinic. Nevertheless, these laboratory approaches are mandatory and constitute a first filter to discriminate among potential drug candidates and chemical compounds that should be discarded.