The in vivo measurement of replication fork velocity and pausing by lag-time analysis

Huang, Dean; Johnson, Anna E.; Sim, Brandon S.; Lo, Teresa; Merrikh, Houra; Wiggins, Paul A.

doi:10.1038/s41467-023-37456-2

Cited by 13 publications

(21 citation statements)

References 56 publications

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“…The fits of the E.coli data reveal that the Peclet number characterizing the replisome dynamics is rather large. On the one hand, this observation confirms that simpler approaches that neglect stochasticity [22,23] provide reliable results, at least in the case of wild type E.coli. On the other hand, the diffusion constant, albeit small, provides important information about the uncertainty of the replisome meeting point.…”

Section: Discussionsupporting

confidence: 73%

“…coli data reveal that the Peclet number characterizing the replisome dynamics is rather large. On the one hand, this observation confirms that simpler approaches that neglect stochasticity [22, 23] provide reliable results, at least in the case of wild type E . coli .…”

Section: Discussionsupporting

confidence: 72%

“…It follows from Eqs. (3) and(4) that Solving for the speed, we obtain i.e., the replication speed is inversely proportional to the logarithmic slope of 𝒫( x ) [22, 23]. An advantage of the deterministic assumption is that it leads to a one-to-one correspondence between DNA abundance and local replisome speed thanks to Eq.…”

Section: Introductionmentioning

confidence: 99%

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Genome replication in asynchronously growing microbial populations

Pflug,

Bhat,

Pigolotti

2023

Preprint

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Biological cells adopt specific programs to replicate their genomes. Information about the replication program of an organism can be obtained by sequencing an exponentially growing cell culture and studying the frequency of DNA fragments as a function of genomic position. However, a quantitative interpretation of this data has been challenging for asynchronously growing cultures. In this paper, we introduce a general theory to predict the abundance of DNA fragments in asynchronously growing cultures from any given stochastic model of the DNA replication program. As key examples, we present stochastic models of DNA replication in Escherichia coli and in budding yeast. In both cases, our approach leads to analytical predictions that are in excellent agreement with experimental data and permit to infer biophysically relevant parameters. In particular, our method is able to infer the locations of known replication origins in budding yeast with high accuracy. These examples demonstrate that our method can provide insight into a broad range of organisms, from bacteria to eukaryotes.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionsupporting

confidence: 72%

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Genome replication in asynchronously growing microbial populations

Pflug,

Bhat,

Pigolotti

2023

Preprint

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“…Furthermore, more insightful information may be within reach based on new high-throughput data and mathematical modeling (Huang et al, 2023; Pflug et al, 2023; Bhat et al, 2023; Le Treut et al, 2021), which can reveal the relationship between initiation and replication dynamics.…”

Section: Discussionmentioning

confidence: 99%

Robust control of replication initiation in the absence of DnaA-ATP ↔ DnaA-ADP regulatory elements inEscherichia coli

Boesen

Charbon

et al. 2022

Preprint

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Replication initiation, especially the widely conserved master initiator protein DnaA, is one of the most well-studied biological problems in bacteria. Specifically, conversion between the ATP and ADP form of DnaA during growth is critical for initiation control. In Escherichia coli, the regulatory elements for conversion have been discovered and extensively characterized over decades. However, they are not widely conserved in bacteria, raising questions on how to generalize the findings in E. coli to other organisms. In this work, we show that the intrinsic ATPase activity of DnaA itself is sufficient for robust and precise initiation control. We constructed and studied E. coli mutants lacking the extrinsic control of either DnaA-ATP → DnaA-ADP (by hda and datA) or DnaA-ADP → DnaA-ATP (by DARS1 and DARS2) at the single-cell and population levels. These cells showed distinct and opposing characteristics in initiation timing, the degree of initiation asynchrony, and cell-to-cell variability. Strikingly, when all four regulatory elements were deleted, E. coli exhibited a near wild-type phenotype, with only mildly increased intrinsic and extrinsic initiation noise. By further characterizing the DnaA variants with increased and decreased ATPase activity, we conclude that DnaA is the only requirement for robust initiation, shedding new evolutionary light on cell-cycle control in bacteria.

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“…Values of C have been estimated using many modes (e.g. 7,[15][16][17] ; some reviewed in 14 ), only two of which are direct, in vivo 19,20 . The uncertainties in values of C, the amazingly low CV of W/NC (Table 1), and the small number of parameters (C and τ only) involved in calculating NC, led us, serendipitously so, to assess if it is possible to obtain C by finding the minimal CV value of W/NC from the available measured data sets (τ, W).…”

mentioning

confidence: 99%

Cell Width inEscherichia coli: Interrogating Coupling to Nucleoid Structure

Howard

Rabinovitch

Yehezkel

et al. 2023

Preprint

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Cell dimensions of rod-shaped bacteria such as Escherichia coli are connected to mass growth and chromosome replication. It divides 20 min after termination of the replication cycle that initiates 40 min earlier at a relatively constant mass. Cells enlarge by elongation only, but at faster growth in richer media they are also wider. Width determination occurs in the divisome during the division process, coupled, temporally and spatially, to the ratio between the rates of growth and replication. The elusive signal directing the mechanism for width determination is related to the tightly linked duplications of the nucleoid (DNA) and the sacculus (peptidoglycan), the only two structures (macro-molecules) existing in a single copy. Six biologically meaningful parameters related to the key number of replication positions are reasonable candidates to convey such a signal. The current analysis discovered that of these, nucleoid complexity is the most likely parameter affecting cell width. As a corollary, a new, indirect approach to estimate replication rate is revealed.

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The in vivo measurement of replication fork velocity and pausing by lag-time analysis

Cited by 13 publications

References 56 publications

Genome replication in asynchronously growing microbial populations

Genome replication in asynchronously growing microbial populations

Robust control of replication initiation in the absence of DnaA-ATP ↔ DnaA-ADP regulatory elements inEscherichia coli

Cell Width inEscherichia coli: Interrogating Coupling to Nucleoid Structure

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