Proliferating cell nuclear antigen (PCNA) is required for mismatch repair (MMR) and has been shown to interact with complexes containing Msh2p or MLH1 (refs 1-4). PCNA has been implicated to act in MMR before and during the DNA synthesis step, although the biochemical basis for the role of PCNA early in MMR is unclear. Here we observe an interaction between PCNA and Msh2p-Msh6p mediated by a specific PCNA-binding site present in Msh6p. An msh6 mutation that eliminated the PCNA-binding site caused a mutator phenotype and a defect in the interaction with PCNA. The association of PCNA with Msh2p-Msh6p stimulated the preferential binding of Msh2p-Msh6p to DNA containing mispaired bases. Mutant PCNA proteins encoded by MMR-defective pol30 alleles were defective for interaction with Msh2p-Msh6p and for stimulation of mispair binding by Msh2p-Msh6p. Our results suggest that PCNA functions directly in mispair recognition and that mispair recognition requires a higher-order complex containing proteins in addition to Msh2p-Msh6p.
The Bacillus subtilis global regulator AbrB is a DNAbinding protein composed of six identical monomers of 96 amino acids that shows specificity to the promoter regions of its target genes including its own. We have sequenced thirteen previously uncharacterized abrB mutations. Four mutant AbrB proteins were purified, and their DNA-binding properties and multimeric structures were examined. AbrB23 (R25S) had no appreciable DNA binding activity but retained a hexameric structure, indicating that Arg25 is important in DNA interactions. Three other mutant proteins, AbrB1 (C56Y), AbrB19 (Gln 83 3 termination codon), and AbrB100 (L69P), showed decreased DNA binding and altered multimeric interactions. Analysis of the expression and AbrB binding affinities of mutant abrB promoters demonstrated that a consensus ؊35 region is incompatible with proper autoregulation of the abrB gene.AbrB is a DNA-binding transcriptional regulator of numerous genes that commence expression at the end of vegetative growth and the onset of stationary phase and sporulation (1, 2). Its primary function is to prevent inappropriate expression of nutrient limitation-induced functions at times when they are not needed. The regulatory role of AbrB is not limited to events associated with growth cessation because it also modulates expression of some genes during slow exponential growth in suboptimal environments. In at least two cases, histidine utilization (3) and ribose transport (4), this involves antagonism of the catabolite repression response caused by slowly metabolizable sugars.AbrB does not share significant amino acid homology with other known classes of DNA-binding proteins (5, 6). It is a hexamer of identical small subunits (96 amino acids), and it appears that the integrity of the hexameric structure is critical for DNA binding (7). DNaseI footprinting on over 20 chromosomal targets has revealed defined protection regions ranging in size from 24 to over 100 contiguous base pairs (3, 4, 8 -11). 1 Examination of these regions has failed to identify a simple DNA consensus sequence that can be unequivocally assigned as a recognition determinant (11). It is believed that AbrB recognizes a specific three-dimensional DNA structure that can be assumed by a finite subset of differing base sequences (1,8,11,12). Using in vitro selection of random oligonucleotides, we have isolated seemingly optimal AbrB-binding sites that possess two to four occurrences of a 5-bp 2 motif (TGGNA) separated by defined spacings (12). However, multiple regularly spaced examples of this motif are rare in chromosomal binding sites. It is thought that such optimal sites are infrequently used in vivo because they are incompatible with promoter structures and with the mechanism responsible for rapid relief of repressive effects due to AbrB-DNA interactions.Mutations in the promoter and coding regions of abrB have been isolated, and a few of them have been sequenced and characterized (7,(13)(14)(15)(16)(17)(18)(19). In this communication, we report the sequence of additiona...
The nonspecific alkaline phosphatase of Saccharomyces sp. strain 1710 has been shown by phosphatase cytochemistry to be exclusively located in the vacuole, para-Nitrophenyl phosphate-specific alkaline phosphatase is not detected by this procedure because the activity of this enzyme is sensitive to the fixative agent, glutaraldehyde. To determine whether the oligosaccharide of nonspecific alkaline phosphatase is necessary to transport the enzyme into the vacuole, protoplasts were derepressed in the absence or in the presence of tunicamycin, an antibiotic which interferes with the glycosylation of asparagine residues in proteins. The location of the enzyme in the tunicamycin-treated protoplasts, as determined by electron microscopy and subcellular fractionation, was identical to its location in control protoplasts. In addition, carbohydrate-free alkaline phosphatase was found in vacuoles from tunicamycin-treated protoplasts. Our findings indicate that the asparagine-linked carbohydrate moiety does not determine the cellular location of the enzyme.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.