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Cell Jan 2023In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands....
In eukaryotes, DNA replication initiation requires assembly and activation of the minichromosome maintenance (MCM) 2-7 double hexamer (DH) to melt origin DNA strands. However, the mechanism for this initial melting is unknown. Here, we report a 2.59-Å cryo-electron microscopy structure of the human MCM-DH (hMCM-DH), also known as the pre-replication complex. In this structure, the hMCM-DH with a constricted central channel untwists and stretches the DNA strands such that almost a half turn of the bound duplex DNA is distorted with 1 base pair completely separated, generating an initial open structure (IOS) at the hexamer junction. Disturbing the IOS inhibits DH formation and replication initiation. Mapping of hMCM-DH footprints indicates that IOSs are distributed across the genome in large clusters aligning well with initiation zones designed for stochastic origin firing. This work unravels an intrinsic mechanism that couples DH formation with initial DNA melting to license replication initiation in human cells.
Topics: Humans; Cell Cycle Proteins; Cryoelectron Microscopy; DNA Replication; DNA-Binding Proteins; Minichromosome Maintenance Proteins; Replication Origin
PubMed: 36608662
DOI: 10.1016/j.cell.2022.12.008 -
Biochemical and Biophysical Research... Dec 2022Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the... (Review)
Review
Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.
Topics: Replication Origin; DNA Replication; Escherichia coli; DNA-Binding Proteins; Replicon; Bacterial Proteins; Chromosomes, Bacterial; DNA, Bacterial
PubMed: 36344169
DOI: 10.1016/j.bbrc.2022.09.060 -
DNA Repair Sep 2022The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by... (Review)
Review
The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by the three major replicases, DNA polymerases α, δ and ε. When an incorrect base is occasionally inserted, the latter Pols δ and ε also have a 3 ´ to 5 ´ exonuclease activity that can remove the mismatch to allow correct DNA synthesis to proceed. Lastly, rare mismatches that escape proofreading activity and are present in newly replicated DNA can be removed by DNA mismatch repair. In this review, we consider evidence supporting the hypothesis that the second mechanism, proofreading, can operate in two different ways. Primer terminal mismatches made by either Pol δ or Pol ε can be 'intrinsically' proofread. This mechanism occurs by direct transfer of a misinserted base made at the polymerase active site to the exonuclease active site that is located a short distance away. Intrinsic proofreading allows mismatch excision without intervening enzyme dissociation. Alternatively, considerable evidence suggests that mismatches made by any of the three replicases can also be proofread by 'extrinsic' proofreading by Pol δ. Extrinsic proofreading occurs when a mismatch made by any of the three replicases is initially abandoned, thereby allowing the exonuclease active site of Pol δ to bind directly to and remove the mismatch before replication continues. Here we review the evidence that extrinsic proofreading significantly enhances the fidelity of nuclear DNA replication, and we then briefly consider the implications of this process for evolution and disease.
Topics: DNA; DNA Polymerase II; DNA Polymerase III; DNA Replication; Exonucleases
PubMed: 35850061
DOI: 10.1016/j.dnarep.2022.103369 -
Microbiology Spectrum Feb 2015Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its... (Review)
Review
Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.
Topics: Archaea; Bacteria; DNA Helicases; DNA Replication; DNA-Directed DNA Polymerase; Models, Biological; Plasmids; Trans-Activators
PubMed: 26104557
DOI: 10.1128/microbiolspec.PLAS-0035-2014 -
Advances in Experimental Medicine and... 2017In eukaryotes, genome duplication starts concomitantly at many replication initiation sites termed replication origins. The replication initiation program is spatially... (Review)
Review
In eukaryotes, genome duplication starts concomitantly at many replication initiation sites termed replication origins. The replication initiation program is spatially and temporally coordinated to ensure accurate, efficient DNA synthesis that duplicates the entire genome while maintaining other chromatin-dependent functions. Unlike in prokaryotes, not all potential replication origins in eukaryotes are needed for complete genome duplication during each cell cycle. Instead, eukaryotic cells vary the use of initiation sites so that only a fraction of potential replication origins initiate replication each cell cycle. Flexibility in origin choice allows each eukaryotic cell type to utilize different initiation sites, corresponding to unique nuclear DNA packaging patterns. These patterns coordinate replication with gene expression and chromatin condensation. Budding yeast replication origins share a consensus sequence that marks potential initiation sites. Metazoan origins, on the other hand, lack a consensus sequence. Rather, they are associated with a collection of structural features, chromatin packaging features, histone modifications, transcription, and DNA-DNA/DNA-protein interactions. These features confer cell type-specific replication and expression and play an essential role in maintaining genomic stability.
Topics: Animals; Chromatin; DNA Replication; Eukaryota; Eukaryotic Cells; Genomic Instability; Humans; Replication Origin; Saccharomycetales
PubMed: 29357052
DOI: 10.1007/978-981-10-6955-0_2 -
Molecular Cell Aug 2023During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during...
During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
Topics: Humans; DNA Primase; Cryoelectron Microscopy; DNA Replication; DNA Helicases; Saccharomyces cerevisiae; DNA, Single-Stranded
PubMed: 37506699
DOI: 10.1016/j.molcel.2023.06.035 -
Microbiology Spectrum Dec 2014Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge... (Review)
Review
Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge and understanding of the structure of iteron-containing replicons, the structure of the iteron plasmid encoded replication initiation proteins, and the molecular mechanisms for iteron plasmid DNA replication initiation. We also discuss the current understanding of control mechanisms affecting the plasmid copy number and how host chaperone proteins and proteases can affect plasmid maintenance in bacterial cells.
Topics: DNA Replication; DNA, Bacterial; Plasmids; Repetitive Sequences, Nucleic Acid
PubMed: 26104462
DOI: 10.1128/microbiolspec.PLAS-0026-2014 -
Journal of Bacteriology Jul 2017In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome... (Review)
Review
In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed , where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways , and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability.
Topics: Bacteria; DNA Repair; DNA Replication; DNA, Bacterial; Mutation
PubMed: 28320884
DOI: 10.1128/JB.00102-17 -
DNA Repair Jul 2021In every cell cycle, billions of nucleotides need to be duplicated within hours, with extraordinary precision and accuracy. The molecular mechanism by which cells... (Review)
Review
In every cell cycle, billions of nucleotides need to be duplicated within hours, with extraordinary precision and accuracy. The molecular mechanism by which cells regulate the replication event is very complicated, and the entire process begins way before the onset of S phase. During the G1 phase of the cell cycle, cells prepare by assembling essential replication factors to establish the pre-replicative complex at origins, sites that dictate where replication would initiate during S phase. During S phase, the replication process is tightly coupled with the DNA repair system to ensure the fidelity of replication. Defects in replication and any error must be recognized by DNA damage response and checkpoint signaling pathways in order to halt the cell cycle before cells are allowed to divide. The coordination of these processes throughout the cell cycle is therefore critical to achieve genomic integrity and prevent diseases. In this review, we focus on the current understanding of how the replication initiation events are regulated to achieve genome stability.
Topics: Animals; Cell Cycle; DNA Replication; Eukaryota; Genomic Instability; Humans; Replication Origin
PubMed: 33992866
DOI: 10.1016/j.dnarep.2021.103131 -
Molecular Cell Jan 2024Genome damage and transcription are intimately linked. Tens to hundreds of thousands of DNA lesions arise in each cell each day, many of which can directly or indirectly... (Review)
Review
Genome damage and transcription are intimately linked. Tens to hundreds of thousands of DNA lesions arise in each cell each day, many of which can directly or indirectly impede transcription. Conversely, the process of gene expression is itself a source of endogenous DNA lesions as a result of the susceptibility of single-stranded DNA to damage, conflicts with the DNA replication machinery, and engagement by cells of topoisomerases and base excision repair enzymes to regulate the initiation and progression of gene transcription. Although such processes are tightly regulated and normally accurate, on occasion, they can become abortive and leave behind DNA breaks that can drive genome rearrangements, instability, or cell death.
Topics: Humans; DNA Damage; DNA Replication; DNA Repair; DNA; Genome; Genomic Instability; Transcription, Genetic
PubMed: 38103560
DOI: 10.1016/j.molcel.2023.11.014