The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future.

The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analog by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k pol ) were generally higher for the variant, the presence of a lesion in the templating position reduced the ability of both the wild type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG•dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.Most replicative DNA polymerases copy DNA with high fidelity. These faithful enzymes must select the correct dNTP among the four canonical nucleotides and do so at rates high enough to support replication of the genome within the time frame of a single cell division. The error rates of replicative DNA polymerases range as high as one error for every 10 6 incorporation events (1). While DNA polymerases have the ability to efficiently discriminate between correct and incorrect incoming nucleoside triphosphates, the genomes of all organisms are under constant assault from both endogenous and exogenous sources. The resulting alterations to the genome can have a dramatic effect on the ability of DNA polymerases to maintain accurate DNA replication or to maintain any replication at all (2). Abasic sites are produced at a rate of >10,000 per human cell per day (3). These lesions arise primarily through spontaneous hydrolysis of the N-glycosylic bond and as intermediates in the base excision repair pathway (4). Abasic sites (Figure 1) are strong blocks to DNA replication by most replicative DNA polymerases and can only be bypassed at a low rate in vitro in the presence of high dNTP concentrations. Under these conditions, all replicative polymerases tested to date exhibit a strong preference for incorporation of dAMP, regardless of the nature of the original templating base. This phenomenon, known as the A-rule, is mutagenic (5,6).* to whom correspondence should be addressed swallace@uvm.edu or sdoublie@uvm.edu tel: 802-656-2164 fax: 802-656-8749. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 23. Published in final edited form as:Biochemistry. Reactive oxygen species pose another significant threat to DNA. In particular, 1 is a commonly formed oxidatively derived lesion that has ...

a b s t r a c tTargeting of Saccharomyces cerevisiae Cdc24p to polarized growth sites is essential for its function. Localization of GFP-tagged Cdc24 proteins or fragments was assayed in deletion mutants of Cdc24p-interacting proteins. The boi2D, ent2D, and hua1D mutants showed localization defects. The tos2D skg6D double mutant displayed aberrant pre-anaphase localization to the mother-bud neck region. The same aberrant pattern was seen when potential phosphorylation sites Ser697, Thr704, and Tyr200 were mutated. The S697A mutation also resulted in phosphorylation defects in vivo. These data support roles for Boi2p, Ent2p, Hua1p, Tos2p, and for Cdc24p phosphorylation in targeting Cdc24p to growth sites.

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