scholarly journals Inversions with deletions and duplications.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 411-414 ◽  
Author(s):  
A J Gordon ◽  
J A Halliday
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
De Novo ◽  
Leading Strand ◽  
Lagging Strand

Abstract Complex mutational events, including de novo inversion with deletion and duplication of sequence, have been observed but are difficult to model. We propose that nascent leading-strand misalignment upon the lagging-strand template during DNA replication can result in the inversion of sequence. The positioning of this misalignment and of the realignment of the leading strand back into the leading-strand template will determine if the inversion is accompanied by deletion and duplication of sequence. We suggest that such strand misalignment-realignment events may occur at the replication fork during concurrent DNA replication.

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 Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

2018 ◽  
Author(s):  
Sarina Y. Porcella ◽  
Natasha C. Koussa ◽  
Colin P. Tang ◽  
Daphne N. Kramer ◽  
Priyanka Srivastava ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Leading Strand ◽  
Strand Initiation ◽  
Lagging Strand

AbstractDuring eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.Author summaryHalf of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

scholarly journals Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

2019 ◽  
Vol 116 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Glen E. Cronan ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Chromosomal Dna ◽  
E Coli ◽  
Okazaki Fragments ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

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 No support for the adaptive hypothesis of lagging-strand encoding in bacterial genomes

2020 ◽  
Author(s):  
Haoxuan Liu ◽  
Jianzhi Zhang
Keyword(s):  
Dna Replication ◽  
Comparative Genomics ◽  
Dna Polymerases ◽  
Purifying Selection ◽  
Deleterious Mutations ◽  
Bacterial Genomes ◽  
Highly Expressed Genes ◽  
Balance Hypothesis ◽  
Leading Strand ◽  
Lagging Strand

ABSTRACTGenes are preferentially encoded on the leading instead of the lagging strand of DNA replication in most bacterial genomes1. This bias likely results from selection against lagging-strand encoding, which can cause head-on collisions between DNA polymerases and RNA polymerases that induce transcriptional abortion, replication delay, and possibly mutagenesis1. But there are still genes encoded on the lagging strand, an observation that has been explained by a balance between deleterious mutations bringing genes from the leading to the lagging strand and purifying selection purging such mutations2. This mutation-selection balance hypothesis predicts that the probability that a gene is encoded on the lagging strand decreases with the detriment of its lagging-strand encoding relative to leading-strand encoding, explaining why highly expressed genes and essential genes are underrepresented on the lagging strand3,4. In a recent study, Merrikh and Merrikh proposed that the observed lagging-strand encoding is adaptive instead of detrimental, due to beneficial mutations brought by the potentially increased mutagenesis resulting from head-on collisions5. They reported empirical observations from comparative genomics that were purported to support their hypothesis5. Here we point out methodological flaws and errors in their analyses and logical problems of their interpretation. Our reanalysis of their data finds no evidence for the adaptive hypothesis.

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.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

Sign in / Sign up

Export Citation Format

Share Document