The haloarchaeal chromosome replication machinery

2009 ◽  
Vol 37 (1) ◽  
pp. 108-113 ◽  
Author(s):  
Stuart A. MacNeill
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Biochemical Analysis ◽  
Structure And Function ◽  
Haloferax Volcanii ◽  
Eukaryotic Cells ◽  
Replication Machinery ◽  
Chromosomal Dna ◽  
Biochemical Studies ◽  
And Function

The powerful combination of genetic and biochemical analysis has provided many key insights into the structure and function of the chromosomal DNA replication machineries of bacterial and eukaryotic cells. In contrast, in the archaea, biochemical studies have dominated, mainly due to the absence of efficient genetic systems for these organisms. This situation is changing, however, and, in this regard, the genetically tractable haloarchaea Haloferax volcanii and Halobacterium sp. NRC-1 are emerging as key models. In the present review, I give an overview of the components of the replication machinery in the haloarchaea, with particular emphasis on the protein factors presumed to travel with the replication fork.

Structure and function of the GINS complex, a key component of the eukaryotic replisome

2010 ◽  
Vol 425 (3) ◽  
pp. 489-500 ◽  
Author(s):  
Stuart A. MacNeill
Keyword(s):  
Replication Fork ◽  
Biochemical Analysis ◽  
Current Knowledge ◽  
Chromosomal Dna ◽  
Minichromosome Maintenance ◽  
Replication Process ◽  
Replicative Helicase ◽  
Cellular Life ◽  
Mcm Helicase ◽  
And Function

High-fidelity chromosomal DNA replication is fundamental to all forms of cellular life and requires the complex interplay of a wide variety of essential and non-essential protein factors in a spatially and temporally co-ordinated manner. In eukaryotes, the GINS complex (from the Japanese go-ichi-ni-san meaning 5-1-2-3, after the four related subunits of the complex Sld5, Psf1, Psf2 and Psf3) was recently identified as a novel factor essential for both the initiation and elongation stages of the replication process. Biochemical analysis has placed GINS at the heart of the eukaryotic replication apparatus as a component of the CMG [Cdc45–MCM (minichromosome maintenance) helicase–GINS] complex that most likely serves as the replicative helicase, unwinding duplex DNA ahead of the moving replication fork. GINS homologues are found in the archaea and have been shown to interact directly with the MCM helicase and with primase, suggesting a central role for the complex in archaeal chromosome replication also. The present review summarizes current knowledge of the structure, function and evolution of the GINS complex in eukaryotes and archaea, discusses possible functions of the GINS complex and highlights recent results that point to possible regulation of GINS function in response to DNA damage.

scholarly journals The encapsulin from Thermatoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein

2021 ◽  
Author(s):  
Benjamin J LaFrance ◽  
Caleb Cassidy-Amstutz ◽  
Robert J Nichols ◽  
Luke M Oltrogge ◽  
Eva Nogales ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Biochemical Analysis ◽  
Active Role ◽  
Structure And Function ◽  
Redox Active ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
And Function ◽  
Flavin Binding ◽  
Unique Chemical

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based "organelles" found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model T. maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryoEM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the T. maritima encapsulin, the decameric cargo protein with 5-fold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme.

scholarly journals THE DNA REPLICATION FORK IN EUKARYOTIC CELLS

1998 ◽  
Vol 67 (1) ◽  
pp. 721-751 ◽  
Author(s):  
Shou Waga ◽  
Bruce Stillman
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Eukaryotic Cells

scholarly journals Unwinding 20 Years of the Archaeal Minichromosome Maintenance Helicase

2020 ◽  
Vol 202 (6) ◽  
Author(s):  
Lori M. Kelman ◽  
William B. O’Dell ◽  
Zvi Kelman
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Duplex Dna ◽  
Chromosomal Dna ◽  
Minichromosome Maintenance ◽  
Replicative Helicase ◽  
Dna Helicases ◽  
First Report ◽  
20Th Anniversary

ABSTRACT Replicative DNA helicases are essential cellular enzymes that unwind duplex DNA in front of the replication fork during chromosomal DNA replication. Replicative helicases were discovered, beginning in the 1970s, in bacteria, bacteriophages, viruses, and eukarya, and, in the mid-1990s, in archaea. This year marks the 20th anniversary of the first report on the archaeal replicative helicase, the minichromosome maintenance (MCM) protein. This minireview summarizes 2 decades of work on the archaeal MCM.

scholarly journals Two essential replicative DNA polymerases exchange dynamically during DNA replication and after replication fork arrest

2018 ◽  
Author(s):  
Yilai Li ◽  
Ziyuan Chen ◽  
Lindsay A. Matthews ◽  
Lyle A. Simmons ◽  
Julie S. Biteen
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Super Resolution ◽  
Clamp Loader ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Replication Process ◽  
Cooperative Process

AbstractThe replisome is the multi-protein complex responsible for faithful replication of chromosomal DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in liveBacillus subtiliscells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following both damage-independent and damage-dependent replication fork stress. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replisomal proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process, and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results show that DnaE provides a limited contribution to chromosomal replication and repair.

scholarly journals Structural insights into the architecture and assembly of eukaryotic flagella

2020 ◽  
Vol 7 (11) ◽  
pp. 289-299
Author(s):  
Narcis-Adrian Petriman ◽  
Esben Lorentzen
Keyword(s):  
Structure And Function ◽  
Eukaryotic Cells ◽  
Repetitive Nature ◽  
Unicellular Organisms ◽  
Cilia And Flagella ◽  
And Function ◽  
Signaling Components ◽  
Structural Insights ◽  
Insight Into

Cilia and flagella are slender projections found on most eukaryotic cells including unicellular organisms such as Chlamydomonas, Trypanosoma and Tetrahymena, where they serve motility and signaling functions. The cilium is a large molecular machine consisting of hundreds of different proteins that are trafficked into the organelle to organize a repetitive microtubule-based axoneme. Several recent studies took advantage of improved cryo-EM methodology to unravel the high-resolution structures of ciliary complexes. These include the recently reported purification and structure determination of axonemal doublet microtubules from the green algae Chlamydomonas reinhardtii, which allows for the modeling of more than 30 associated protein factors to provide deep molecular insight into the architecture and repetitive nature of doublet microtubules. In addition, we will review several recent contributions that dissect the structure and function of ciliary trafficking complexes that ferry structural and signaling components between the cell body and the cilium organelle.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin J. LaFrance ◽  
Caleb Cassidy-Amstutz ◽  
Robert J. Nichols ◽  
Luke M. Oltrogge ◽  
Eva Nogales ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Thermotoga Maritima ◽  
Active Role ◽  
Structure And Function ◽  
Redox Active ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
And Function ◽  
Flavin Binding ◽  
Unique Chemical

AbstractBacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based ‘organelles’ found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25–50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure–function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier—it also plays a significant role in the structure and function of the cargo enzyme.

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