Replication dynamics of recombination-dependent replication forks

AbstractReplication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ. Here, we use polymerase usage sequencing to visualize in vivo replication dynamics of HR-restarted forks 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.

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Replication dynamics of recombination-dependent replication forks

10.1101/2020.07.03.186676 ◽

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.

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Faculty Opinions recommendation of Defining fractional inhibitory concentration index cutoffs for additive interactions based on self-drug additive combinations, Monte Carlo simulation analysis, and in vitro-in vivo correlation data for antifungal drug combinations against Aspergillus fumigatus.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1282045.749142 ◽

2009 ◽

Author(s):

Annette Fothergill

Keyword(s):

Monte Carlo Simulation ◽

Monte Carlo ◽

Concentration Index ◽

Inhibitory Concentration ◽

Simulation Analysis ◽

Fractional Inhibitory Concentration ◽

Fractional Inhibitory Concentration Index ◽

Correlation Data

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Study of the Influence of Radionuclide Biokinetics on the Efficiency of In Vivo Counting Using Monte Carlo Simulation

Health Physics ◽

10.1097/01.hp.0000342828.21935.e4 ◽

2009 ◽

Vol 96 (5) ◽

pp. 558-567 ◽

Cited By ~ 11

Author(s):

Stephanie Lamart ◽

Eric Blanchardon ◽

Andrey Molokanov ◽

Gary H. Kramer ◽

David Broggio ◽

...

Keyword(s):

Monte Carlo Simulation ◽

Monte Carlo

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SU-E-J-145: Validation of An Analytical Model for in Vivo Range Verification Using GATE Monte Carlo Simulation in Proton Therapy

Medical Physics ◽

10.1118/1.4924230 ◽

2015 ◽

Vol 42 (6Part9) ◽

pp. 3297-3298

Author(s):

C Lee ◽

H Lin ◽

T Chao ◽

I Hsiao ◽

K Chuang

Keyword(s):

Monte Carlo Simulation ◽

Monte Carlo ◽

Analytical Model ◽

Proton Therapy ◽

Range Verification

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Monte Carlo simulation of near infrared autofluorescence measurements of in vivo skin

Journal of Photochemistry and Photobiology B Biology ◽

10.1016/j.jphotobiol.2011.08.008 ◽

2011 ◽

Vol 105 (3) ◽

pp. 183-189 ◽

Cited By ~ 13

Author(s):

Shuang Wang ◽

Jianhua Zhao ◽

Harvey Lui ◽

Qingli He ◽

Haishan Zeng

Keyword(s):

Monte Carlo Simulation ◽

Monte Carlo ◽

Near Infrared

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814512116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1251-1260 ◽

Cited By ~ 7

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.

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