DNA Replication

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (31 March 2015) | Viewed by 123162

Special Issue Editor

Department of Medicine, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, A-1090 Wien, Austria
Interests: enzymology of DNA replication and repair; RNA metabolism; molecular evolution of nucleases

Special Issue Information

Dear Colleagues,

Replication of DNA, even of the simplest one, is a very sophisticated process, depending on a complex enzymatic machinery being evolved over more than a billion of years. The proper action of replication is a prerequisite to maintain the genome integrity of all living organisms and together with other mechanisms it enables the evolution of life. On the one hand the universality of basic principles of duplication, derived from data obtained from the prokaryote kingdom of life, is an undisputed fact in our times. On the other hand the coordination of replication of eukaryotic genomes and the interaction between duplication and the cell division cycle is still a matter of intense research and debate. This special issue intends to demonstrate new developments and concepts in the general field of DNA replication and related topics, e.g. DNA repair. Research or review articles on any topic within the field will be considered. We are looking forward to your contributions.

Prof. Dr. Peter Frank 
Guest Editor

Keywords

  • eukaryotic DNA replication
  • replication fork dynamics
  • origin
  • priming
  • Okazaki fragments
  • DNA polymerases
  • nucleases
  • topoisomerases
  • helicases

Published Papers (10 papers)

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Research

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1103 KiB  
Article
Arrest of Viral Proliferation by Ectopic Copies of Its Cognate Replication Origin
by Manuel S. Valenzuela and Chakradhari Sharan
Genes 2015, 6(2), 436-450; https://doi.org/10.3390/genes6020436 - 23 Jun 2015
Cited by 26 | Viewed by 4559
Abstract
The initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the [...] Read more.
The initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the presence of ectopic origin copies may interfere with proper genome duplication. Bacteriophage λ was used as a model system. To this end, the outcome of an infection of an E. coli strain harboring ectopic copies of the λ origin region was analyzed. By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication. These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation. Full article
(This article belongs to the Special Issue DNA Replication)
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433 KiB  
Article
An Inactive Geminin Mutant That Binds Cdt1
by Marissa Suchyta, Benoit Miotto and Thomas J. McGarry
Genes 2015, 6(2), 252-266; https://doi.org/10.3390/genes6020252 - 15 May 2015
Cited by 8 | Viewed by 6011
Abstract
The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. [...] Read more.
The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7), the replication helicase, into the pre-replication complex (pre-RC) at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1. Full article
(This article belongs to the Special Issue DNA Replication)
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1285 KiB  
Article
Trapping DNA Replication Origins from the Human Genome
by Toshihiko Eki, Yasufumi Murakami and Fumio Hanaoka
Genes 2013, 4(2), 198-225; https://doi.org/10.3390/genes4020198 - 17 Apr 2013
Cited by 25 | Viewed by 9215
Abstract
Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using [...] Read more.
Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks. Full article
(This article belongs to the Special Issue DNA Replication)
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407 KiB  
Article
A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C
by Ankita Chiraniya, Jeff Finkelstein, Mike O'Donnell and Linda B. Bloom
Genes 2013, 4(2), 134-151; https://doi.org/10.3390/genes4020134 - 26 Mar 2013
Cited by 6 | Viewed by 8212
Abstract
In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker [...] Read more.
In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker A and Walker B sequence motifs, which participate in ATP binding and hydrolysis, respectively. Mutation of the glutamate residue in Walker B motifs (or DExx-boxes) in AAA+ proteins typically reduces ATP hydrolysis by as much as a couple orders of magnitude, but has no effect on ATP binding. Here, the Walker B Glu in each of the four active ATP sites of the eukaryotic clamp loader, RFC, was mutated to Gln and Ala separately, and ATP binding- and hydrolysis-dependent activities of the quadruple mutant clamp loaders were characterized. Fluorescence-based assays were used to measure individual reaction steps required for clamp loading including clamp binding, clamp opening, DNA binding and ATP hydrolysis. Our results show that the Walker B mutations affect ATP-binding-dependent interactions of RFC with the clamp and DNA in addition to reducing ligand-dependent ATP hydrolysis activity. Here, we show that the Walker B glutamate is required for ATP-dependent ligand binding activity, a previously unknown function for this conserved Glu residue in RFC. Full article
(This article belongs to the Special Issue DNA Replication)
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Review

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297 KiB  
Review
Regulation of Unperturbed DNA Replication by Ubiquitylation
by Sara Priego Moreno and Agnieszka Gambus
Genes 2015, 6(3), 451-468; https://doi.org/10.3390/genes6030451 - 25 Jun 2015
Cited by 15 | Viewed by 7329
Abstract
Posttranslational modification of proteins by means of attachment of a small globular protein ubiquitin (i.e., ubiquitylation) represents one of the most abundant and versatile mechanisms of protein regulation employed by eukaryotic cells. Ubiquitylation influences almost every cellular process and its key [...] Read more.
Posttranslational modification of proteins by means of attachment of a small globular protein ubiquitin (i.e., ubiquitylation) represents one of the most abundant and versatile mechanisms of protein regulation employed by eukaryotic cells. Ubiquitylation influences almost every cellular process and its key role in coordination of the DNA damage response is well established. In this review we focus, however, on the ways ubiquitylation controls the process of unperturbed DNA replication. We summarise the accumulated knowledge showing the leading role of ubiquitin driven protein degradation in setting up conditions favourable for replication origin licensing and S-phase entry. Importantly, we also present the emerging major role of ubiquitylation in coordination of the active DNA replication process: preventing re-replication, regulating the progression of DNA replication forks, chromatin re-establishment and disassembly of the replisome at the termination of replication forks. Full article
(This article belongs to the Special Issue DNA Replication)
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227 KiB  
Review
Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III
by Hiroshi Arakawa and George Iliakis
Genes 2015, 6(2), 385-398; https://doi.org/10.3390/genes6020385 - 23 Jun 2015
Cited by 19 | Viewed by 7550
Abstract
Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present [...] Read more.
Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a universal DNA ligase, which can potentially substitute or backup the repair and replication functions of all other DNA ligases in the cell nucleus. Thus, the old model of functionally dedicated DNA ligases is now replaced by one in which only Lig4 remains dedicated to C-NHEJ, with Lig1 and Lig3 showing an astounding functional flexibility and interchangeability for practically all nuclear ligation functions. The underlying mechanisms of Lig3 versus Lig1 utilization in DNA repair and replication are expected to be partly different and remain to be elucidated. Full article
(This article belongs to the Special Issue DNA Replication)
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1362 KiB  
Review
Replication Stress in Mammalian Cells and Its Consequences for Mitosis
by Camille Gelot, Indiana Magdalou and Bernard S. Lopez
Genes 2015, 6(2), 267-298; https://doi.org/10.3390/genes6020267 - 22 May 2015
Cited by 79 | Viewed by 15022
Abstract
The faithful transmission of genetic information to daughter cells is central to maintaining genomic stability and relies on the accurate and complete duplication of genetic material during each cell cycle. However, the genome is routinely exposed to endogenous and exogenous stresses that can [...] Read more.
The faithful transmission of genetic information to daughter cells is central to maintaining genomic stability and relies on the accurate and complete duplication of genetic material during each cell cycle. However, the genome is routinely exposed to endogenous and exogenous stresses that can impede the progression of replication. Such replication stress can be an early cause of cancer or initiate senescence. Replication stress, which primarily occurs during S phase, results in consequences during mitosis, jeopardizing chromosome segregation and, in turn, genomic stability. The traces of replication stress can be detected in the daughter cells during G1 phase. Alterations in mitosis occur in two types: 1) local alterations that correspond to breaks, rearrangements, intertwined DNA molecules or non-separated sister chromatids that are confined to the region of the replication dysfunction; 2) genome-wide chromosome segregation resulting from centrosome amplification (although centrosomes do not contain DNA), which amplifies the local replication stress to the entire genome. Here, we discuss the endogenous causes of replication perturbations, the mechanisms of replication fork restart and the consequences for mitosis, chromosome segregation and genomic stability. Full article
(This article belongs to the Special Issue DNA Replication)
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2237 KiB  
Review
Replication Checkpoint: Tuning and Coordination of Replication Forks in S Phase
by Nicole Hustedt, Susan M. Gasser and Kenji Shimada
Genes 2013, 4(3), 388-434; https://doi.org/10.3390/genes4030388 - 19 Aug 2013
Cited by 44 | Viewed by 12155
Abstract
Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. During S phase, cells also [...] Read more.
Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. During S phase, cells also provide a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication forks. The DNA replication checkpoint is crucial for maintaining genome stability, because replication forks become vulnerable to collapse when they encounter obstacles such as nucleotide adducts, nicks, RNA-DNA hybrids, or stable protein-DNA complexes. These can be exogenously induced or can arise from endogenous cellular activity. Here, we summarize the initiation and transduction of the replication checkpoint as well as its targets, which coordinate cell cycle events and DNA replication fork stability. Full article
(This article belongs to the Special Issue DNA Replication)
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681 KiB  
Review
The Replication Fork: Understanding the Eukaryotic Replication Machinery and the Challenges to Genome Duplication
by Adam R. Leman and Eishi Noguchi
Genes 2013, 4(1), 1-32; https://doi.org/10.3390/genes4010001 - 29 Jan 2013
Cited by 69 | Viewed by 44978
Abstract
Eukaryotic cells must accurately and efficiently duplicate their genomes during each round of the cell cycle. Multiple linear chromosomes, an abundance of regulatory elements, and chromosome packaging are all challenges that the eukaryotic DNA replication machinery must successfully overcome. The replication machinery, the [...] Read more.
Eukaryotic cells must accurately and efficiently duplicate their genomes during each round of the cell cycle. Multiple linear chromosomes, an abundance of regulatory elements, and chromosome packaging are all challenges that the eukaryotic DNA replication machinery must successfully overcome. The replication machinery, the “replisome” complex, is composed of many specialized proteins with functions in supporting replication by DNA polymerases. Efficient replisome progression relies on tight coordination between the various factors of the replisome. Further, replisome progression must occur on less than ideal templates at various genomic loci. Here, we describe the functions of the major replisome components, as well as some of the obstacles to efficient DNA replication that the replisome confronts. Together, this review summarizes current understanding of the vastly complicated task of replicating eukaryotic DNA. Full article
(This article belongs to the Special Issue DNA Replication)
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Other

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5512 KiB  
Concept Paper
Modeling of the SV40 DNA Replication Machine
by Daniel T. Simmons
Genes 2012, 3(4), 742-758; https://doi.org/10.3390/genes3040742 - 09 Nov 2012
Cited by 28 | Viewed by 6465
Abstract
The mechanism of SV40 DNA replication is certainly not completely understood. The proteins that are necessary for replication have been known for quite some time, but how they work together to form a nanomachine capable of faithfully replicating the virus DNA is only [...] Read more.
The mechanism of SV40 DNA replication is certainly not completely understood. The proteins that are necessary for replication have been known for quite some time, but how they work together to form a nanomachine capable of faithfully replicating the virus DNA is only partially understood. Some of the proteins involved have been crystallized and their 3D structures determined, and several EM reconstructions of SV40 T antigen have been generated. In addition, there is a fair amount of biochemical data that pinpoints the sites of interaction between various proteins. With this information, various models were assembled that show how the SV40 DNA replication nanomachine could be structured in three dimensional space. This process was aided by the use of a 3D docking program as well as fitting of structures. The advantage of the availability of these models is that they are experimentally testable and they provide an insight into how the replication machine could work. Another advantage is that it is possible to quickly compare newly published structures to the models in order to come up with improved models. Full article
(This article belongs to the Special Issue DNA Replication)
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