Modular Structure of the Human Lamin B2 Replicator

ABSTRACT The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Download Full-text

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

10.1101/2021.03.01.433423 ◽

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.

Download Full-text

DNA replication origins retain mobile licensing proteins

Nature Communications ◽

10.1038/s41467-021-22216-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Humberto Sánchez ◽

Kaley McCluskey ◽

Theo van Laar ◽

Edo van Veen ◽

Filip M. Asscher ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Origin Recognition Complex ◽

Atp Hydrolysis ◽

Protein Diffusion ◽

Mcm Helicase ◽

Slow Moving ◽

Dna Replication Origins ◽

Mobile Protein ◽

Origin Recognition

AbstractDNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.

Download Full-text

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006231117 ◽

2020 ◽

Vol 117 (30) ◽

pp. 17747-17756 ◽

Cited By ~ 2

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.

Download Full-text

A predictable conserved DNA base composition signature defines human core DNA replication origins

Nature Communications ◽

10.1038/s41467-020-18527-0 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Ildem Akerman ◽

Bahar Kasaai ◽

Alina Bazarova ◽

Pau Biak Sang ◽

Isabelle Peiffer ◽

...

Keyword(s):

Dna Replication ◽

Dna Sequence ◽

Cell Types ◽

Replication Initiation ◽

Replication Origins ◽

Dna Base Composition ◽

Dna Replication Initiation ◽

Sequence Elements ◽

Dna Replication Origins ◽

Genomic Regions

Abstract DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.

Download Full-text

Identification andin silicoanalysis of the origin recognition complex in the human fungal pathogenCandida albicans

10.1101/430892 ◽

2018 ◽

Author(s):

Sreedevi Padmanabhan ◽

Kaustuv Sanyal ◽

Dharani Dhar Dubey

Keyword(s):

Dna Replication ◽

Origin Recognition Complex ◽

3D Structure ◽

Domain Architecture ◽

Model Organism ◽

Replication Origins ◽

Replication Machinery ◽

Cellular Processes ◽

Dna Replication Origins ◽

Origin Recognition

AbstractDNA replication in eukaryotes is initiated by the orchestrated assembly and association of initiator proteins (heterohexameric Origin Recognition Complex, ORC) on the replication origins. These functionally conserved proteins play significant roles in diverse cellular processes besides their central role in ignition of DNA replication at origins. WhileCandida albicans, a major human fungal pathogen, is an ascomycetous, asexual, diploid budding yeast but it is significantly diverged from a much better studied model organismSaccharomyces cerevisiae. The components of the DNA replication machinery inC. albicansremain largely uncharacterized. Identification of factors required for DNA replication is essential for understanding the evolution of the DNA replication machinery. We identified the putative ORC homologs inC. albicansand determined their relatedness with those of other eukaryotes including several yeast species. Our extensivein silicostudies demonstrate that the domain architecture of CaORC proteins share similarities with the ORC proteins ofS. cerevisiae. We dissect the domain organization of ORC (trans-acting factors) proteins that seem to associate with DNA replication origins inC. albicans. We present a model of the 3D structure of CaORC4 to gain further insights of this protein’s function.

Download Full-text

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

Genes & Development ◽

10.1101/gad.14.13.1631 ◽

2000 ◽

Vol 14 (13) ◽

pp. 1631-1641 ◽

Cited By ~ 1

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.

Download Full-text