scholarly journals Structures and operating principles of the replisome

Science ◽  
2019 ◽  
Vol 363 (6429) ◽  
pp. eaav7003 ◽  
Author(s):  
Yang Gao ◽  
Yanxiang Cui ◽  
Tara Fox ◽  
Shiqiang Lin ◽  
Huaibin Wang ◽  
...  
Keyword(s):  
Replication Fork ◽  
Model System ◽  
Molecular Organization ◽  
Cryo Electron Microscopy ◽  
Adenosine Triphosphatases ◽  
Dna Strands ◽  
Leading Strand ◽  
Dna Strand ◽  
Aaa Atpases ◽  
Lagging Strand

Visualization in atomic detail of the replisome that performs concerted leading– and lagging–DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo–electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

scholarly journals 3′ → 5′ Exonucleases of DNA Polymerases ϵ and δ Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 717-726 ◽  
Author(s):  
Polina V Shcherbakova ◽  
Youri I Pavlov
Keyword(s):  
Dna Replication ◽  
Dna Polymerases ◽  
Replication Errors ◽  
Dna Strands ◽  
Base Analog ◽  
Chromosome Iii ◽  
Dna Strand ◽  
Dna Replication Errors ◽  
Yeast Mutants ◽  
Lagging Strand

Abstract The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC → AT and AT → GC transitions that are enhanced in DNA polymerase ϵ and δ 3′ → 5′ exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ϵ contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC → AT and AT → GC transitions in a Pol→, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ϵ and δ correct HAP-induced DNA replication errors on opposite DNA strands.

scholarly journals Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

2016 ◽  
Vol 113 (21) ◽  
pp. 5916-5921 ◽  
Author(s):  
Alfredo J. Hernandez ◽  
Seung-Joo Lee ◽  
Charles C. Richardson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Fragment Length ◽  
Dna Binding Protein ◽  
Okazaki Fragment ◽  
Dna Primase ◽  
Dna Strands ◽  
Leading Strand ◽  
Rate Limiting ◽  
Lagging Strand

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

scholarly journals Human mismatch repair system corrects errors produced during lagging strand replication more effectively

2016 ◽  
Author(s):  
Maria Andrianova ◽  
Georgii A Bazykin ◽  
Sergey Nikolaev ◽  
Vladimir Seplyarskiy
Keyword(s):  
Mismatch Repair ◽  
Mutation Rates ◽  
Repair System ◽  
Wild Type ◽  
Strand Asymmetry ◽  
Exonuclease Domain ◽  
Mismatch Repair System ◽  
Dna Strands ◽  
Leading Strand ◽  
Lagging Strand

Mismatch repair (MMR) is one of the main systems maintaining fidelity of replication. Different effectiveness in correction of errors produced during replication of the leading and the lagging DNA strands was reported in yeast, but this effect is poorly studied in humans. Here, we use MMR-deficient (MSI) and MMR-proficient (MSS) cancer samples to investigate properties of the human MMR. MSI, but not MSS, cancers demonstrate unequal mutation rates between the leading and the lagging strands. The direction of strand asymmetry in MSI cancers matches that observed in cancers with mutated exonuclease domain of polymerase δ, indicating that polymerase δ contributes more mutations than its leading-strand counterpart, polymerase ε. As polymerase δ primarily synthesizes DNA during the lagging strand replication, this implies that mutations produced in wild type cells during lagging strand replication are repaired by the MMR ~3 times more effectively, compared to those produced on the leading strand.

scholarly journals The CMG helicase bypasses DNA protein cross-links to facilitate their repair

2018 ◽  
Author(s):  
Justin L. Sparks ◽  
Alan O. Gao ◽  
Markus Räschle ◽  
Nicolai B. Larsen ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Replication Fork ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Cross Link ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Cross Links ◽  
Leading Strand ◽  
Nucleoprotein Complexes ◽  
Lagging Strand

SummaryCovalent and non-covalent nucleoprotein complexes impede replication fork progression and thereby threaten genome integrity. UsingXenopus laevisegg extracts, we previously showed that when a replication fork encounters a covalent DNA-protein cross-link (DPC) on the leading strand template, the DPC is degraded to a short peptide, allowing its bypass by translesion synthesis polymerases. Strikingly, we show here that when DPC proteolysis is blocked, the replicative DNA helicase (CMG), which travels on the leading strand template, still bypasses the intact DPC. The DNA helicase RTEL1 facilitates bypass, apparently by translocating along the lagging strand template and generating single-stranded DNA downstream of the DPC. Remarkably, RTEL1 is required for efficient DPC proteolysis, suggesting that CMG bypass of a DPC normally precedes its proteolysis. RTEL1 also promotes fork progression past non-covalent protein-DNA complexes. Our data suggest a unified model for the replisome’s response to nucleoprotein barriers.

scholarly journals Replication dynamics of recombination-dependent replication forks

2020 ◽  
Author(s):  
Karel Naiman ◽  
Eduard Campillo-Funollet ◽  
Adam T. Watson ◽  
Alice Budden ◽  
Izumi Miyabe ◽  
...  
Keyword(s):  
Monte Carlo Simulation ◽  
Monte Carlo ◽  
Homologous Recombination ◽  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Leading Strand ◽  
The Stability ◽  
Lagging Strand

AbstractDNA replication fidelity is essential for maintaining genetic stability. Forks arrested at replication fork barriers can be stabilised by the intra-S phase checkpoint, subsequently being rescued by a converging fork, or resuming when the barrier is removed. However, some arrested forks cannot be stabilised and fork convergence cannot rescue in all situations. Thus, cells have developed homologous recombination-dependent mechanisms to restart persistently inactive forks. To understand HR-restart we use polymerase usage sequencing to visualize in vivo replication dynamics at an S. pombe replication barrier, RTS1, and model replication by Monte Carlo simulation. We show that HR-restarted forks synthesise both strands with Pol δ for up to 30 kb without maturing to a δ/ε configuration and that Pol α is not used significantly on either strand, suggesting the lagging strand template remains as a gap that is filled in by Pol δ later. We further demonstrate that HR-restarted forks progress uninterrupted through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, signifying the stability of the 3’ single strand in the context of increased resection.

scholarly journals Replisome bypass of transcription complexes and R-loops

2020 ◽  
Vol 48 (18) ◽  
pp. 10353-10367
Author(s):  
Jan-Gert Brüning ◽  
Kenneth J Marians
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Instability ◽  
Bacterial Dna ◽  
Template Strand ◽  
Replication Fork Progression ◽  
Leading Strand ◽  
Fork Stalling ◽  
Transcription Complexes ◽  
Lagging Strand

Abstract The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.

scholarly journals DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3489
Author(s):  
Youri I. Pavlov ◽  
Anna S. Zhuk ◽  
Elena I. Stepchenkova
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Dna Polymerases ◽  
Structure And Function ◽  
The Past ◽  
New Findings ◽  
Dna Strands ◽  
Leading Strand ◽  
And Function

Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization. The principal idea of participation of different polymerases in specific transactions at the fork proposed by Morrison and coauthors 30 years ago and later named “division of labor,” remains standing, with an amendment of the broader role of polymerase δ in the replication of both the lagging and leading DNA strands. However, cancer-associated mutations predominantly affect the catalytic subunit of polymerase ε that participates in leading strand DNA synthesis. We analyze how new findings in the DNA replication field help elucidate the polymerase variants’ effects on cancer.

Chromatin replication

2019 ◽  
pp. 165-172
Author(s):  
John C. Lucchesi
Keyword(s):  
Dna Replication ◽  
The Other ◽  
Replication Process ◽  
Dna Molecule ◽  
Origins Of Replication ◽  
Dna Strands ◽  
Leading Strand ◽  
Fork Stalling ◽  
Nucleosomal Structure ◽  
Lagging Strand

During chromosome replication, each strand of the DNA serves as a template for the synthesis of a new strand (semiconservative replication). Replication originates with the binding of pre-replication complexes (MCM2–7) at multiple sites, termed origins of replication (ORIs). One strand of the parent DNA molecule (the leading strand) is replicated continuously in the 5´ to 3´ direction; the other, or lagging strand, is replicated one short segment at a time. These segments are referred to as Okazaki fragments, and their generation requires the synthesis of short complementary RNA primers. For replication to proceed, DNA must be unwound and freed from nucleosomes. After replication, the nucleosomal structure is re-established using a mixture of old and newly synthesized histones. DNA replication can encounter problems such as nucleotide depletion, DNA damage or topologically unfavorable structures that generate replication stress and replication fork stalling. The DNA replication process itself can also be subjected to errors. Cells have evolved a battery of mechanisms designed to bypass or repair damaged DNA strands.

scholarly journals Architecture of the Replication Fork Stalled at the 3′ End of Yeast Ribosomal Genes

2000 ◽  
Vol 20 (15) ◽  
pp. 5777-5787 ◽  
Author(s):  
Markus Gruber ◽  
Ralf Erik Wellinger ◽  
José M. Sogo
Keyword(s):  
Replication Fork ◽  
Rrna Gene ◽  
Replication Forks ◽  
Branched Dna ◽  
Unique Site ◽  
Leading Strand ◽  
Nucleotide Resolution ◽  
Endonuclease Vii ◽  
First Time ◽  
Lagging Strand

ABSTRACT Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within anHpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found. In the majority of molecules, the stalled lagging strand was completely processed and the discontinuously synthesized nascent lagging strand was extended three bases farther than the continuously synthesized leading strand. A model explaining these findings is presented. Our analysis included for the first time the use of T4 endonuclease VII, an enzyme recognizing branched DNA molecules. This enzyme cleaved predominantly in the newly synthesized homologous arms, thereby specifically releasing the leading arm.

scholarly journals Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

2017 ◽  
Vol 114 (10) ◽  
pp. E1848-E1856 ◽  
Author(s):  
Arkadiusz W. Kulczyk ◽  
Arne Moeller ◽  
Peter Meyer ◽  
Piotr Sliz ◽  
Charles C. Richardson
Keyword(s):  
Replication Fork ◽  
Molecular Mechanisms ◽  
Dna Polymerases ◽  
Spatial Arrangement ◽  
Dna Helicase ◽  
Cryo Electron Microscopy ◽  
Replication Systems ◽  
Processivity Factor ◽  
Lagging Strand ◽  
Multiple Interactions

We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading- and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading- and lagging-strand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.

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