Biomedical subjects

Justin J Donato

Publications and source records attributed to Justin J Donato.

5 recordsLinked to original sources

PubMed2006

Genome-wide hierarchy of replication origin usage in Saccharomyces cerevisiae.

Replication origins in a genome are inherently different in their base sequence and in their response to temporal and cell cycle regulation signals for DNA replication. To investigate the chromosomal determinants that influence the efficiency of initiation of DNA replication genome-wide, we made use of a reverse strategy originally used for the isolation of replication initiation mutants in Saccharomyces cerevisiae. In yeast, replication origins isolated from chromosomes support the autonomous replication of plasmids. These replication origins, whether in the context of a chromosome or a plasmid, will initiate efficiently in wild-type cells but show a dramatically contrasted efficiency of activation in mutants defective in the early steps of replication initiation. Serial passages of a genomic library of autonomously replicating sequences (ARSs) in such a mutant allowed us to select for constitutively active ARSs. We found a hierarchy of preferential initiation of ARSs that correlates with local transcription patterns. This preferential usage is enhanced in mutants defective in the assembly of the prereplication complex (pre-RC) but not in mutants defective in the activation of the pre-RC. Our findings are consistent with an interference of local transcription with the assembly of the pre-RC at a majority of replication origins.

DNA, Fungal↗

PubMed2005

Dual roles for Mcm10 in DNA replication initiation and silencing at the mating-type loci.

Recent studies linking DNA replication proteins to transcriptional silencing suggest that some of the same mechanisms that facilitate the initiation of replication at origins might be involved in establishing repressed chromatin at silencer elements. Our ongoing studies of several mutants of the replication initiation factor Mcm10 of budding yeast revealed an associated defect in the production of mating type pheromones. This observation prompted us to look more directly at the effect of MCM10 mutations on the expression of a reporter gene in the mating type locus and to assay for physical interactions between Mcm10 and known silencing factors. Our findings, that Mcm10 mutants disrupt mating loci silencing and that Mcm10 interacts with Sir2 and Sir3, suggest that Mcm10 also plays an essential, and separable role in transcriptional silencing.

Cell Cycle Proteins↗

PubMed2004

Mcm1 promotes replication initiation by binding specific elements at replication origins.

Minichromosome maintenance protein 1 (Mcm1) is required for efficient replication of autonomously replicating sequence (ARS)-containing plasmids in yeast cells. Reduced DNA binding activity in the Mcm1-1 mutant protein (P97L) results in selective initiation of a subset of replication origins and causes instability of ARS-containing plasmids. This plasmid instability in the mcm1-1 mutant can be overcome for a subset of ARSs by the inclusion of flanking sequences. Previous work showed that Mcm1 binds sequences flanking the minimal functional domains of ARSs. Here, we dissected two conserved telomeric X ARSs, ARS120 (XARS6L) and ARS131a (XARS7R), that replicate with different efficiencies in the mcm1-1 mutant. We found that additional Mcm1 binding sites in the C domain of ARS120 that are missing in ARS131a are responsible for efficient replication of ARS120 in the mcm1-1 mutant. Mutating a conserved Mcm1 binding site in the C domain diminished replication efficiency in ARS120 in wild-type cells, and increasing the number of Mcm1 binding sites stimulated replication efficiency. Our results suggest that threshold occupancy of Mcm1 in the C domain of telomeric ARSs is required for efficient initiation. We propose that origin usage in Saccharomyces cerevisiae may be regulated by the occupancy of Mcm1 at replication origins.

Base Sequence↗

PubMed2003

Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1.

The Saccharomyces cerevisiae Mcm7 protein is a subunit of the presumed heteromeric MCM helicase that melts origin DNA and unwinds replication forks. Previous work showed that Mcm1 binds constitutively to the MCM7 promoter and regulates MCM7 expression. Here, we identify Mcm7 as a novel cofactor of Mcm1 in the regulation of MCM7 expression. Transcription of MCM7 is increased in the mcm7-1 mutant and decreased in the mcm1-1 mutant, suggesting that Mcm7 modulates its own expression in conjunction with Mcm1. Indeed, Mcm7 stimulates Mcm1 binding to the early cell cycle box upstream of the promoters of MCM7 as well as CDC6 and MCM5. Whereas Mcm1 binds these promoters constitutively, Mcm7 is recruited during late M phase, consistent with Mcm7 playing a direct role in modulating the periodic expression of early cell cycle genes. The multiple roles of Mcm7 in replication initiation, replication elongation, and autoregulation parallel those of the oncoprotein, the large T-antigen of the SV40 virus.

Antigens, Polyomavirus Transforming↗

PubMed2002

Mcm1 binds replication origins.

Mcm1 is an essential protein required for the efficient replication of minichromosomes and the transcriptional regulation of early cell cycle genes in Saccharomyces cerevisiae. In this study, we report that Mcm1 is an abundant protein that associates globally with chromatin in a punctate pattern. We show that Mcm1 is localized at replication origins and plays an important role in the initiation of DNA synthesis at a chromosomal replication origin in vivo. Using purified Mcm1 protein, we show that Mcm1 binds cooperatively to multiple sites at autonomously replicating sequences. These results suggest that, in addition to its role as a transcription factor for the expression of replication genes, Mcm1 may influence the local structure of replication origins by direct binding.

Base Sequence↗