Biology subjects

Hollatz, A. J.

Publications and source records attributed to Hollatz, A. J..

3 recordsLinked to original sources

bioRxiv2024

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes the G1-chromatin status and activity of chromosomal DNA replication origins

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of 100 origins that act in early to mid-S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

molecular biology↗

bioRxiv2021

The Fkh1 Forkhead Associated Domain Promotes ORC Binding to a Subset of DNA Replication Origins in Budding Yeast

The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognition complex (ORC), which directs the formation of origins, the specific chromosomal regions where DNA will be unwound for the initiation of DNA synthesis. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (forkhead homolog) protein, and, in particular, its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. The majority of the FHA-dependent origins within the experimental subset examined contain a distinct Fkh1 binding site located 5 of and proximal to their ORC sites (5-FKH-T site). Epistasis experiments using selected FHA-dependent origins provided evidence that the FHA domain promoted origin activity through Fkh1 binding directly to this 5 FKH-T site. Nucleotide substitutions within two of these origins that enhanced the affinity of their ORC sites for ORC bypassed these origins requirement for their 5 FKH-T sites and for the FHA domain. Significantly, direct assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism affected ~25% of yeast origins. Thus, this study reveals a new mechanism to enhance ORC-origin binding in budding yeast that requires the FHA domain of the conserved cell-cycle transcription factor Fkh1.

molecular biology↗

bioRxiv2019

A Meier-Gorlin Syndrome Mutation in Orc4 CausesTissue-Specific DNA Replication Defects in Drosophila melanogaster

Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct developmental defects, including primordial dwarfism, small ears, and small or missing patella. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, and MCM5) are mutated in individuals diagnosed with MGS. The primary role of ORC is to license origins during the G1 phase of the cell cycle, but it also plays roles in cilia development, heterochromatin formation, and other cellular processes. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in ORC lead to the tissue-specific defects associated with the disease. To address this question, we have used Cas9-mediated genome engineering to generate a Drosophila melanogaster model of individuals carrying a mutation in ORC4. Like the people with Meier-Gorlin syndrome, these flies reach adulthood, but have several tissue-specific defects. Genetic analysis revealed that this allele is a hypomorph and that mutant females are sterile. We demonstrated that this sterility is caused by a failure in DNA replication. By leveraging the well-studied Drosophila system, we showed that a disease-causing mutation in orc4 disrupts DNA replication, and we propose that in individuals with MGS defects arise preferentially in tissues with a high-replication demand.

developmental biology↗