Unwinding 20 Years of the Archaeal Minichromosome Maintenance Helicase

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.

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

Biochemical Journal ◽

10.1042/bj20091531 ◽

2010 ◽

Vol 425 (3) ◽

pp. 489-500 ◽

Cited By ~ 68

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.

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How is the archaeal MCM helicase assembled at the origin? Possible mechanisms

Biochemical Society Transactions ◽

10.1042/bst0370007 ◽

2009 ◽

Vol 37 (1) ◽

pp. 7-11 ◽

Cited By ~ 9

Author(s):

Nozomi Sakakibara ◽

Lori M. Kelman ◽

Zvi Kelman

Keyword(s):

Replication Fork ◽

Biochemical Properties ◽

Duplex Dna ◽

Origin Of Replication ◽

Minichromosome Maintenance ◽

Replicative Helicase ◽

Loading Process ◽

Mcm Helicase

In order for any organism to replicate its DNA, a helicase must unwind the duplex DNA in front of the replication fork. In archaea, the replicative helicase is the MCM (minichromosome maintenance) helicase. Although much is known about the biochemical properties of the MCM helicase, the mechanism of assembly at the origin of replication is unknown. In the present paper, several possible mechanisms for the loading process are described.

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

10.1101/364695 ◽

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.

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Nuclease dead Cas9 is a programmable roadblock for DNA replication

10.1101/455543 ◽

2018 ◽

Cited By ~ 1

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.

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Replication Fork Uncoupling Causes Nascent Strand Degradation and Fork Reversal

10.1101/2021.10.13.464136 ◽

2021 ◽

Author(s):

Tamar Kavlashvili ◽

James M Dewar

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Genome Stability ◽

Replicative Helicase ◽

Before And After ◽

Egg Extracts ◽

Xenopus Egg ◽

Biochemical Approach ◽

Key Steps ◽

Xenopus Egg Extracts

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. The replicative helicase remains bound during NSD and fork reversal, indicating that both processes take place behind the helicase. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and identify key steps involved in these processes.

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The haloarchaeal chromosome replication machinery

Biochemical Society Transactions ◽

10.1042/bst0370108 ◽

2009 ◽

Vol 37 (1) ◽

pp. 108-113 ◽

Cited By ~ 14

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.

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Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes

Science China Life Sciences ◽

10.1007/s11427-014-4631-4 ◽

2014 ◽

Vol 57 (5) ◽

pp. 482-487 ◽

Cited By ~ 10

Author(s):

LiHong Wu ◽

Yang Liu ◽

DaoChun Kong

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Initiation ◽

Chromosomal Dna ◽

Dna Replication Initiation

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Xenopus Cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading

Genes & Development ◽

10.1101/gad.14.12.1528 ◽

2000 ◽

Vol 14 (12) ◽

pp. 1528-1540

Author(s):

Pedro Jares ◽

J. Julian Blow

Keyword(s):

Dna Replication ◽

Origin Recognition Complex ◽

Protein Complexes ◽

Present Report ◽

S Phase ◽

Chromosomal Dna ◽

Minichromosome Maintenance ◽

Initiation Of Replication ◽

Sequential Binding

The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication. The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin. Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase. In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs. We show that XCdc7 binds to chromatin during G1 and S phase. XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing. Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete. Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function. From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system.

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Overlap of replication rounds disturbs the progression of replicating forks in a ribonucleotide reductase mutant of Escherichia coli

Microbiology ◽

10.1099/mic.0.047316-0 ◽

2011 ◽

Vol 157 (7) ◽

pp. 1955-1967 ◽

Cited By ~ 3

Author(s):

Israel Salguero ◽

Elena López Acedo ◽

Elena C. Guzmán

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Ribonucleotide Reductase ◽

Replication Fork ◽

Rna Synthesis ◽

Restrictive Temperature ◽

Protein Synthesis Inhibition ◽

Chromosomal Dna ◽

Dnaa Protein ◽

Density Results

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, Escherichia coli cells carrying the nrdA101 allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 °C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 °C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 °C cannot explain the completion of chromosomal DNA replication in the nrdA101 mutant. We found that inactivation of the DnaA protein by using several dnaAts alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the nrdA101 mutant at 42 °C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies of datA sequence increases the relative amount of DNA synthesis of the nrdA101 mutant at 42 °C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.

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