Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.

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Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.Chromosomal origins of DNA replication in budding yeast are called autonomously replicating sequence (ARS) elements and were identified about 30 years ago by their ability to confer autonomous replication to originless plasmids (30). A conserved 11-bp sequence called the ARS consensus sequence (ACS) was initially identified by the analysis of the DNA sequences of four ARS elements (8). The ACS is now known to comprise a binding site for the origin recognition complex (ORC), the essential initiator protein in all eukaryotes (reviewed in reference 2). The consensus sequence of the AT-rich ACS element (WTTTAYRTTTW) is degenerate, and bona fide ARSs sometimes contain only a 10/11-or 9/11-bp match to this sequence. A W indicates the base A or T. When additional ACSs were identified experimentally, a 17-bp extended ACS (EACS) was defined and reflected that the bases flanking the ACS were often A's or T's (WWW-ACS-[G/T]WW) (47). Although the ACS is essential for replicator activity, it cannot be the sole determinant of ORC binding and/or origin specification. By pattern matching, there are 860 exact matches to the 11-bp ACS and 13,978 ACSs, allowing one mismatch in the yeast nuclear genome (http://seq.yeastgenome.org/cgi-bin /PATMATCH/nph-patmatch). Since there are only ϳ350 functional ARS elements in Saccharomyces cerevisiae, excluding the ribosomal DNA locus (25), additional sequences, chromatin environment, active transcription units, higher-order nucleosome structure, and/or nuclear organization might further restrict the locations of functional ARS elements. In agreement with one of these predictions, genome-wide studies of ORC and MCM binding sites revealed that Ͼ90% of putative ARSs are intergenic (50, 51). Furthermore, additional sequences in the B region adjacent to the ACS (Fig. 1) are known to contribute to ORC binding and origin activity in S. cerevisiae (26,37,41,46).Detailed analysis of ARS1 (26) and ARS307 (37, 46) showed that they have modular structures (Fig. 1). In addition to the A...

The inactivation of microorganisms is the most important function in the processing of milk and dairy products. Traditionally, this purpose is realized by thermal treatment, but heat produces alterations to flavor and taste in addition to nutrient loss. The high intensity pulsed electric field (PEF) treatment should be a good alternative to heat because demonstrations have shown PEF can reduce the Escherichia coli survival fraction in aqueous solutions and model foods. In this study, PEF treatment was found to inactivate E. coli in skim milk (inoculum 109 CFU/mL) at 15C. The microorganism inactivation satisfied Hülsheger's model following a first order kinetic for both the electric field intensity and number of pulses when skim milk inoculated with E. coli was treated in a static or continuous flow chamber. PEF treatment in a continuous system when the critical electric field (Ec) and minimum number of pulses (nmin) were 12.34 kV/cm and 2.7 at 30 kV/cm and 30 pulses (0.7–1.8 μs pulse width) inactivated more microorganisms than in a static system. It has also been proven that increasing the pulse duration increases the E. coli inactivation. The inactivation of E. coli using PEF is more limited in skim milk than in a buffer solution when exposed to similar treatment conditions of field intensity and number of pulses due to the complex composition of skim milk, its lower electrical resistivity and the presence of proteins.