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

2021 ◽  
Author(s):  
Timothy Hoggard ◽  
Allison J. Hollatz ◽  
Rachel Cherney ◽  
Catherine A. Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Budding Yeast ◽  
Chromosomal Dna ◽  
Nucleotide Substitutions ◽  
Fha Domain ◽  
Direct Assessment ◽  
Eukaryotic Dna ◽  
Dna Replication Origins

AbstractThe 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.

scholarly journals Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early and late replicating euchromatin

2020 ◽  
Author(s):  
Timothy Hoggard ◽  
Carolin A. Müller ◽  
Conrad A. Nieduszynski ◽  
Michael Weinreich ◽  
Catherine A. Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spatial Distribution ◽  
Dna Replication ◽  
Genome Stability ◽  
Chromatin Modification ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Eukaryotic Chromosome ◽  
Origin Licensing ◽  
Dna Replication Origins

AbstractA eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled on chromosomal DNA in G1-phase (a.k.a. origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing established the normal spatial distribution of origins across yeast chromosomes required for normal genome duplication.Significance statementIn eukaryotes, multiple DNA replication origins, the sites where new DNA synthesis begins during the process of cell division, must be adequately distributed across chromosomes to maintain normal cell proliferation and genome stability. This study describes a repressive chromatin-mediated mechanism that acts at the level of individual origins to attenuate the efficiency of origin formation. This attenuation is essential for achieving the normal spatial distribution of origins across the chromosomes of the eukaryotic microbe Saccharomyces cerevisiae. While the importance of chromosomal origin distribution to cellular fitness is now widely acknowledged, this study is the first to define a specific chromatin modification that establishes the normal spatial distribution of origins across a eukaryotic genome.

scholarly journals A novel role for Cdc5p in DNA replication.

1996 ◽  
Vol 16 (12) ◽  
pp. 6775-6782 ◽  
Author(s):  
C F Hardy ◽  
A Pautz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Budding Yeast ◽  
Yeast Saccharomyces Cerevisiae ◽  
Plasmid Maintenance ◽  
Multiple Origins ◽  
Synthetically Lethal ◽  
Origin Recognition

DNA replication initiates from specific chromosomal sites called origins, and in the budding yeast Saccharomyces cerevisiae these sites are occupied by the origin recognition complex (ORC). Dbf4p is proposed to play a role in targeting the G1/S kinase Cdc7p to initiation complexes late in G1. We report that Dbf4p may also recruit Cdc5p to origin complexes. Cdc5p is a member of the Polo family of kinases that is required for the completion of mitosis. Cdc5p and Cdc7p each interact with a distinct domain of Dbf4p. cdc5-1 mutants have a plasmid maintenance defect that can be suppressed by the addition of multiple origins. cdc5-1 orc2-1 double mutants are synthetically lethal. Levels of Cdc5p were found to be cell cycle regulated and peaked in G2/M. These results suggest a role for Cdc5p and possibly Polo-like kinases at origin complexes.

scholarly journals Conservation of ARS Elements and Chromosomal DNA Replication Origins on Chromosomes III of Saccharomyces cerevisiae and S. carlsbergensis

Genetics ◽  
1999 ◽  
Vol 152 (3) ◽  
pp. 933-941 ◽  
Author(s):  
Chen Yang ◽  
James F Theis ◽  
Carol S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Open Reading Frames ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Brewing Yeast ◽  
Ars Elements ◽  
Intergenic Regions ◽  
Dna Replication Origins ◽  
Chromosome Iii

AbstractDNA replication origins, specified by ARS elements in Saccharomyces cerevisiae, play an essential role in the stable transmission of chromosomes. Little is known about the evolution of ARS elements. We have isolated and characterized ARS elements from a chromosome III recovered from an alloploid Carlsberg brewing yeast that has diverged from its S. cerevisiae homeologue. The positions of seven ARS elements identified in this S. carlsbergensis chromosome are conserved: they are located in intergenic regions flanked by open reading frames homologous to those that flank seven ARS elements of the S. cerevisiae chromosome. The S. carlsbergensis ARS elements were active both in S. cerevisiae and S. monacensis, which has been proposed to be the source of the diverged genome present in brewing yeast. Moreover, their function as chromosomal replication origins correlated strongly with the activity of S. cerevisiae ARS elements, demonstrating the conservation of ARS activity and replication origin function in these two species.

scholarly journals Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6

2020 ◽  
Vol 117 (30) ◽  
pp. 17747-17756 ◽  
Author(s):  
Zuanning Yuan ◽  
Sarah Schneider ◽  
Thomas Dodd ◽  
Alberto Riera ◽  
Lin Bai ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Origin Recognition Complex ◽  
Main Body ◽  
Structural Mechanism ◽  
Helicase Loading ◽  
M Phase ◽  
Dna Insertion ◽  
Eukaryotic Dna ◽  
Dna Replication Origins

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC–Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC–Cdc6–Cdt1–Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC–Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In “semi-attached OCCM,” the Mcm3 and Mcm7 WHDs latch onto ORC–Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In “pre-insertion OCCM,” the main body of Mcm2-7 docks onto ORC–Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2–Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.

scholarly journals Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro

2000 ◽  
Vol 14 (13) ◽  
pp. 1631-1641 ◽  
Author(s):  
Tohru Mizushima ◽  
Naoko Takahashi ◽  
Bruce Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Binding Activity ◽  
Chromosomal Dna ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Dna Replication Origins ◽  
Origin Recognition

An interaction between the origin recognition complex (ORC) and Cdc6p is the first and a key step in the initiation of chromosomal DNA replication. We describe the assembly of an origin-dependent complex containing ORC and Cdc6p from Saccharomyces cerevisiae. Cdc6p increases the DNA binding specificity of ORC by inhibiting non-specific DNA binding of ORC. Cdc6p induces a concomitant change in the conformation of ORC and mutations in the Cdc6p Walker A and Walker B motifs, or ATP-γ-S inhibited these activities of Cdc6p. These data suggest that Cdc6p modifies ORC function at DNA replication origins. On the basis of these results in yeast, we propose that Cdc6p may be an essential determinant of origin specificity in metazoan species.

scholarly journals ORC binds and remodels nucleosomes to specify MCM loading onto DNA

2021 ◽  
Author(s):  
Sai Li ◽  
Michael R. Wasserman ◽  
Olga Yurieva ◽  
Lu Bai ◽  
Michael E. O’Donnell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Sequence ◽  
Single Molecule ◽  
Origin Recognition Complex ◽  
Histone Dimers ◽  
Mcm Helicase ◽  
Eukaryotic Dna ◽  
Higher Eukaryotes ◽  
Origin Function

ABSTRACTSaccharomyces cerevisiae has been a faithful guide for study of eukaryotic DNA replication, as the numerous initiation and elongation proteins are conserved from yeast to human. However, there is a gap in our knowledge of why yeast uses a consensus DNA sequence at replication origins, while higher eukaryotes do not. The current study closes this gap. By direct single-molecule visualization, we show that the Origin Recognition Complex (ORC) searches for and stably binds nucleosomes, and that nucleosomes funtionalize ORC to load MCM helicase onto DNA, regardless of DNA sequence. Furthermore, we discover that ORC can remodel nucleosomes and expel H2A-H2B histone dimers, a heretofore unexpected function. Thus ORC helps create a chromatin environment permissive to origin function. The finding that ORC binding to nucleosomes leads to MCM loading at any DNA sequence is likely to generalize, and that higher eukaryotes follow this same paradigm for origin selection

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

scholarly journals Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

2001 ◽  
Vol 21 (8) ◽  
pp. 2790-2801 ◽  
Author(s):  
James F. Theis ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Binding Site ◽  
Binding Sites ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Discrete Site ◽  
Origin Function ◽  
Single Binding Site ◽  
Origin Recognition

ABSTRACT While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins inSaccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

Method of mapping DNA replication origins

1985 ◽  
Vol 5 (1) ◽  
pp. 85-92
Author(s):  
L D Spotila ◽  
J A Huberman
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Forks ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Dna Restriction Fragments ◽  
Dna Restriction ◽  
Dna Replication Origins

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.

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