Why Can Dna Pol Not Synthesize Lagging Strand Continuously
use an innovative single-molecule imaging approach in yeast cells to measure chromatin association of individual replisome subunits, thereby challenging the notion that lagging-strand DNA polymerases frequently dissociate from replisomes during DNA replication in vivo.
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Within the eukaryotic replisome, the tight coordination of DNA polymerases ensures the coordinated replication of the leading and lagging strands. Once the replicative CMG helicase has unwound the DNA, continuous leading strand synthesis is carried out by polymerase epsilon (Pol ε); on the lagging strand, polymerase alpha (Pol α)-primase synthesizes the RNA primer and a short stretch of DNA, which is extended by polymerase delta (Pol δ). This has conventially been considered an iterative process, requiring both Pol α and Pol δ to cycle in and out of the replisome between the synthesis of each Okazaki fragment (
). Although the division of labor between polymerases has been widely studied, the exact stoichiometry of subcomplexes within the replisome and how stably these complexes associate is less clear.
provide new insights into the behavior of individual replisome proteins by using an elegant in vivo single-molecule imaging approach.
Many recent insights into eukaryotic DNA replication have come from experimental systems using reconstituted yeast replisomes (
). A recent single-molecule study suggested that replisome association of all replicative polymerases was stable, such that the lagging-strand polymerases remained associated for many rounds of Okazaki fragment synthesis (
). However, a limitation of in vitro studies is that they cannot anticipate effects of non-core replisome factors or regulatory events, such as posttranslational modification. However, single-molecule studies of replisomes in living yeast cells are complicated by the presence of ~300 active replisomes during mid-S phase. Moreover, because only about 10% of DNA polymerases are replisome associated (
Saner et al., 2013
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), background signal due to unbound polymerases would be too high to monitor single replisomes (Figure 1A).
circumvent these limitations by labeling only a subset of individual replisome proteins and analyzing their residence time on chromatin. The authors fused HaloTag to individual replisome subunits and added a photoactivatable dye. A short, low dose laser pulse activates only a few molecules, which are tracked via a long image acquisition time that blurs the signal from freely diffusing proteins. Thus, only chromatin-bound molecules form distinct foci across several imaging frames (Figure 1A, right panel): the lifetime of these foci provides an estimate of how long the tagged subunit remains chromatin associated.
Figure 1 Single-Molecule Imaging of Replisomes Reveals Stable Association of Lagging-Strand Polymerases
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(A) Single-molecule imaging of DNA polymerases bound to replisomes is challenged by the high number of active replisomes (~300 in mid-S phase) and the large fraction of unbound DNA polymerase molecules (~90%) that lead to high background signal. The method presented by
relies on HaloTag-labeled polymerase bound to a photoactivatable dye. Upon a short laser pulse, only a few molecules are activated. Repeated excitation and image acquisition with low temporal resolution depict replisome-bound polymerases as distinct foci, whereas soluble polymerases will blur.
(B) Summary of polymerase stability data from
.
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By tracking the residence times of subunits of the three replicative polymerases and using this as a proxy for association with the replisome,
redefine the prevailing notion of replisome dynamics in vivo. As expected, both the CMG helicase itself and the Pol ε subunits that interact with it show long residence times consistent with long-lived association. In contrast, Pol α showed a more dynamic behavior with shorter residence times, albeit still long enough to prime multiple Okazaki fragments before dissociating from the replisome. Most unexpectedly, the Pol δ subunits Pol3 and Pol32 had residence times nearly comparable to Pol ε subunits (Figure 1B). These data contradict the model that a new Pol δ holoenzyme is required for each Okazaki fragment: indeed, the residence times observed suggest that the same Pol δ complex remained associated for many minutes and perhaps even for the entire lifetime of the replisome.
provide additional theoretical support for their experimental model through simulations of free Pol δ rebinding to replisomes within the nucleus. Using biologically plausible parameters, the dissociation of Pol δ after synthesizing an Okazaki fragment and subsequent reassociation with a replisome by diffusion would leave most replisomes lacking Pol δ and therefore unable to efficiently synthesize the lagging strand.
The data by
invite reanalysis of some long-standing observations in eukaryotic replication and open up many future questions. Logically, both Pol α and Pol δ must dissociate from DNA upon completion of one Okazaki fragment, yet both remain bound to chromatin, and thus, presumably at least peripherally associated with the replisome, for more than one round of Okazaki fragment synthesis. What is the nature of the association in between synthesis events? How is polymerase retention ensured, and how is it modulated when replication is perturbed?
Pol δ binds replisomes through both Pol α and the sliding clamp PCNA, yet both of these interacting partners show shorter residence times than Pol δ, suggesting that additional stabilizing contacts must be required. Both Pol ε and Pol α have been shown to directly interact with histones and serve as histone chaperones. Pol α binds to both H3-H4 tetramers and H2A-H2B dimers (
Evrin et al., 2018
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Histone H2A-H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin.
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;
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DNA polymerase a interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands.
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), whereas Pol ε binds parental H3-H4 tetramers (
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A mechanism for preventing asymmetric histone segregation onto replicating DNA strands.
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). An analogous interaction with histones could help retain Pol δ close to the replisome in vivo, although the long-lived association of lagging-strand polymerases in vitro was observed on a non-chromatinized template in the absence of histones (
).
In vitro, the replicative polymerases associate stably to form a processive replisome but can exchange with excess polymerases in solution—a phenomenon known as concentration-dependent exchange (
).
's modeling suggests that cellular concentrations of Pol δ are insufficient for such exchange to occur frequently; however, it is possible that the intermediate stability of Pol ⍺ reflects such exchange. Pol ⍺ levels must be severely reduced to elicit a detectable effect on replication (
Porcella et al., 2020
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Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation.
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), consistent with a substantial excess of Pol ⍺ in the nucleus. However, one of the more surprising observations made by
is that Pol ⍺'s chromatin residence time is unaffected by the absence of Ctf4. Pol ⍺-Ctf4 interaction has previously been shown to be essential for Pol ⍺ to immunoprecipitate with core replisome components but dispensable for efficient replication (
Evrin et al., 2018
- Evrin C.
- Maman J.D.
- Diamante A.
- Pellegrini L.
- Labib K.
Histone H2A-H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin.
- Crossref
- PubMed
- Scopus (34)
- Google Scholar
). Continuous association of DNA polymerases with chromatin might thus be separable from their retention within the replisome itself.
Finally, although it is important to study replisome dynamics in unperturbed conditions, it remains of great interest how such dynamics contribute at stressed replisomes. Prokaryotic replisomes appear to be destabilized by replication-transcription conflicts (
), and such conflicts may be widespread in eukaryotic cells. In yeast, replication across DNA lesions requires translesion synthesis (TLS) polymerases, including Pol ζ, which shares non-catalytic subunits with Pol δ and whose recruitment requires exchange of the non-shared subunits. Moreover, the restart of stalled or broken replication forks is enabled by a high adaptability of replisomes, most likely also involving changes in DNA polymerase contacts with the replisome.
Our understanding of the eukaryotic replisome and its function remains a work in progress. The in vivo single-molecule approach presented by
expands the portfolio of tools available to study this fascinating machine both under optimal conditions and in response to the many challenges encountered during DNA replication.
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DOI: https://doi.org/10.1016/j.molcel.2020.09.010
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- Processive Activity of Replicative DNA Polymerases in the Replisome of Live Eukaryotic Cells
Molecular Cell September 10, 2020
- In Brief
By establishing single-molecule methods in live budding yeast, Kapadia et al. measured the binding kinetics in the eukaryotic replisome. They show that the leading and lagging strand polymerases, Pol δ and Pol ε, are stably bound to the replisome. In contrast, Pol α primase performs few priming cycles before dissociating.
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