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. 2024 Aug 27;52(15):8880-8896.
doi: 10.1093/nar/gkae565.

Single-molecule characterization of SV40 replisome and novel factors: human FPC and Mcm10

Yujing Ouyang  1 , Amani Al-Amodi  1 , Muhammad Tehseen  1 , Lubna Alhudhali  1 , Afnan Shirbini  1 , Masateru Takahashi  1 , Vlad-Stefan Raducanu  1 , Gang Yi  1 , Ammar Usman Danazumi  1 , Alfredo De Biasio  1 , Samir M Hamdan  1
Affiliations

Single-molecule characterization of SV40 replisome and novel factors: human FPC and Mcm10

Yujing Ouyang et al. Nucleic Acids Res. .

Abstract

The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45-MCM-GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Single-molecule characterization of Pol δ primer extension. (A) DNA substrate for primer extension. A primer was annealed to circular M13mp18 ssDNA to introduce Biotin and Digoxigenin at each end separately, with an EcoRI site in the middle. EcoRI restriction enzyme digestion linearized the primer-annealed circular ssDNA to produce a 3′ primer terminus. (B) Representative trace showing Pol δ-dependent primer extension. The black line represents smoothing by a Fast Fourier Transform (FFT) filter. One event's rate, processivity, and lifetime are defined as indicated. (C) Histogram of Pol δ-dependent primer extension apparent rates. The black line represents a Gaussian fit with a rate of 240 ± 30 nt/s (mean ± SEM) (n = 151 events). (D) Histogram of Pol δ-dependent primer extension apparent processivities. The black line represents a single exponential decay fit with a processivity of 0.4 ± 0.1 knt (mean ± SEM) (n = 151 events). All schematics were created with BioRender.com.
Figure 2.
Figure 2.
Single-molecule characterization of L-Tag unwinding. (A) DNA substrate for L-Tag alone, which contains a replication fork at one end of the 13.5-kb dsDNA fragment and a bead attached at the other end. A representative trace shows L-Tag-dependent dsDNA unwinding. The black line represents smoothing by a FFT filter. (B) DNA substrate for L-Tag unwinding with RPA, which contains a replication fork at one end of the 7-kb dsDNA fragment and a bead attached at the same end, at the end of the leading-strand arm of the fork. A representative trace showing L-Tag-dependent dsDNA unwinding with RPA. The black line represents smoothing by a FFT filter. Single-step processivity, pause, multi-step lifetime and multi-step processivity are defined as indicated. (C) Histogram of L-Tag-dependent unwinding apparent rates. The black line represents a Gaussian fit with a rate of 1.1 ± 0.1 bp/s (mean ± SEM) (n = 51 events). (D) Histogram of L-Tag-dependent unwinding apparent rates with RPA. The black line represents a Gaussian fit with a rate of 1.4 ± 0.5 bp/s (mean ± SEM) (n = 81 events). (E) Histogram of L-Tag-dependent unwinding multi-step processivities with RPA. The black line represents a single exponential decay fit with a processivity of 0.8 ± 0.2 kb (mean ± SEM) (n = 40 traces). All schematics were created with BioRender.com.
Figure 3.
Figure 3.
Single-molecule characterization of SV40 leading-strand synthesis. (A) SV40 replisome pre-assembly steps. DNA substrates and beads were tethered in the flow cell, and Pol δ and PCNA were loaded with RFC (not illustrated) at the replication fork. The two dNTPs were added to prevent the exonuclease activity of Pol δ, and L-Tag was loaded. The two dNTPs were still present to prevent Pol δ exonuclease activity. Mg2+ and ATP were removed to prevent L-Tag helicase activity. The following extensive wash retained the same buffer conditions but contained no protein. To begin SV40 leading-strand synthesis, Mg2+, ATP and all four dNTPs are added, including single-strand-binding proteins. (B) The simplified schematic of the DNA substrate in Figure 2A. The representative trace in red shows SV40 replisome-dependent leading-strand synthesis with E. coli SSB compatible with it. The black line represents smoothing by a FFT filter. Single-step processivity, pause, multi-step lifetime and multi-step processivity are defined as indicated. (C) The simplified schematic of the DNA substrate in Figure 2B. The representative trace in grey shows SV40 replisome-dependent leading-strand synthesis with RPA compatible with it. The black line represents smoothing by a FFT filter. (D) Histograms of SV40 replisome-dependent leading-strand synthesis apparent rates. The one with RPA is in grey, fitted by Gaussian distribution with a rate of 4.5 ± 0.4 bp/s (mean ± SEM) (n = 84 events). The one with E. coli SSB is in red, fitted by Gaussian distribution with a rate of 5.3 ± 0.5 bp/s (mean ± SEM) (n = 111 events). (E) Histograms of SV40 replisome-dependent leading-strand synthesis multi-step processivities. The one with RPA is in grey, fitted by single exponential decay distribution with a processivity of 0.5 ± 0.1 kb (mean ± SEM) (n = 39 traces). The one with E. coli SSB is in red, fitted by single exponential decay distribution with a processivity of 0.5 ± 0.1 kb (mean ± SEM) (n = 78 traces). All schematics were created with BioRender.com.
Figure 4.
Figure 4.
FPC and Mcm10 effects in bulk. (A) Schematic of L-Tag unwinding bulk assay and native PAGE gel of L-Tag unwinding products, 5′ Cy5-labelled. The DNA substrate was 0.5 nM. L-Tag was 25 nM. FPC and Mcm10 were 15 nM when indicated. Both FPC and Mcm10 stimulated L-Tag unwinding. (B) Schematic of SV40 leading-strand synthesis bulk assay and alkaline agarose gel of SV40 leading-strand synthesis products. Schematic of the substrate and reaction steps (Left). Product of DNA replication visualized via 32P-dCTP-labelled (nucleotide incorporation). The DNA substrate was 1.5 nM. L-Tag was 30 nM. FPC and Mcm10 were 15 nM when indicated. All schematics and gel labelling were created with BioRender.com.
Figure 5.
Figure 5.
FPC and Mcm10 effects on SV40 leading-strand synthesis in single-molecule. (A) Schematic of SV40 leading-strand synthesis with human FPC and Mcm10. (B) Representative traces showing SV40 leading-strand synthesis (red), SV40 leading-strand synthesis with FPC (olive), SV40 leading-strand synthesis with Mcm10 (blue), and SV40 leading-strand synthesis with FPC and Mcm10 (orange). Black lines represent smoothing by FFT filters. Dashed lines indicate pauses. (C) Histograms of SV40 leading-strand synthesis apparent rates. For SV40 alone, the Gaussian fit gives a mean rate of 5.3 ± 0.5 bp/s (mean ± SEM). The arithmetic average rate is 11 ± 1.1 bp/s (average ± SE) (n = 111 events). For SV40 with FPC, the Gaussian fit gives a mean rate of 3.8 ± 0.8 bp/s (mean ± SEM). The arithmetic average rate is 23 ± 1.9 bp/s (average ± SE) (n = 121 events). For SV40 with Mcm10, the Gaussian fit gives a mean rate of 2.0 ± 1.9 bp/s (mean ± SEM). The arithmetic average rate is 11 ± 1.2 bp/s (average ± SE) (n = 131 events). For SV40 with FPC and Mcm10, the Gaussian fit gives a mean rate of 3.8 ± 0.4 bp/s (mean ± SEM). The arithmetic average rate is 11 ± 0.4 bp/s (average ± SE) (n = 973 events). (D) All statistics of SV40 leading-strand synthesis. Their actual distributions are presented in Supplementary Figures S16, S18, S19, S21–S23. All schematics were created with BioRender.com.
Figure 6.
Figure 6.
Polymerase exchange assays. (A) Schematic: SV40 leading-strand synthesis with polymerase exchange. As DNA replication starts, Pol δ WT is present together with E. coli SSB. Top panel: histogram of SV40 leading-strand synthesis apparent single-step processivities. The black line represents a single exponential decay fit with a processivity of 0.5 ± 0.1 kb (mean ± SEM) (n = 52 traces). Middle panel: histogram of SV40 leading-strand synthesis multi-step processivities. The black line represents a single exponential decay fit with a processivity of 0.6 ± 0.2 kb (mean ± SEM) (n = 43 traces). Lower panel: histogram of SV40 leading-strand synthesis apparent rates has no difference from the normal one. The black line represents a Gaussian fit with a rate of 4.2 ± 0.1 bp/s (mean ± SEM) (n = 52 events). (B) Schematic: SV40 leading-strand synthesis with polymerase exchange. As DNA replication starts, Pol δ polymerase-deficient mutant is present together with E. coli SSB. Top panel: histogram of SV40 leading-strand synthesis apparent single-step processivities. The black line represents a single exponential decay fit with a processivity of 0.6 ± 0.1 kb (mean ± SEM) (n = 96 traces). Middle panel: histogram of SV40 leading-strand synthesis multi-step processivities. The black line represents a single exponential decay fit with a processivity of 0.7 ± 0.1 kb (mean ± SEM) (n = 64 traces). Lower panel: histogram of SV40 leading-strand synthesis apparent rates has no difference from the normal one. The black line represents a Gaussian fit with a rate of 4.2 ± 0.6 bp/s (mean ± SEM) (n = 94 events). (C) Pol δ polymerase-deficient mutant terminates SV40 leading-strand synthesis after certain processivity, time titration. Schematic of Pol δ polymerase-deficient mutant exchange bulk assays and alkaline agarose gels of SV40 leading-strand synthesis products, 32P-dCTP-labelled (nucleotide incorporation). Reactions were performed as described in Materials and methods. All schematics and gel labelling were created with BioRender.com.
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