Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation
- Howard Hughes Medical Institute, Rockefeller University, United States
Figures
Figure 1 with 2 supplements
Pol α primes and extends leading and lagging strands with CMG helicase.
(A) Scheme of the nucleotide-biased 3.2 kb fork substrate used in our experiments, explained further in ‘Materials and methods’. (B) Left: scheme of the assay. CMG is assembled onto the linear 3.2 …
Figure 1—figure supplement 1
SDS-PAGE of purified Pol α.
Pol α (2 μg) was analyzed in a 8% SDS polyacrylamide gel stained with Coomassie Brilliant Blue. The left lane, labeled ‘MM’ represents the Broad Molecular Weight markers (Bio-Rad). Subunits, labeled …
Figure 1—figure supplement 2
Pol α activity on the 3 kb forked DNA is dependent on CMG.
Alkaline gel of omission assays for the Pol α, CMG, RPA leading strand replication reactions illustrated in Figure 1B,C. Lanes 1–6 show a time course of the complete replication reactions for the …
Figure 2
Pol α requires CMG for priming activity during unwinding of forked DNA.
(A) Scheme of assays comparing Pol α activity using either CMG helicase or the strand displacing ϕ29 polymerase. (B) Autoradiograph of DNA products using either 32P-dCTP (leading) or 32P-dGTP …
Figure 3
Okazaki Fragments are produced along the entire DNA.
(A) Restriction enzyme map of the 3.2 kb substrate for Psi I and Ear I. (B) Lagging strand reactions were performed as detailed in ‘Materials and methods’ using an unprimed forked DNA, CMG, RPA, and …
Figure 4 with 3 supplements
Pol ε switches with Pol α on the leading strand but is not active on the lagging strand.
(A) Left: scheme of the assay. Middle: titration of Pol α into leading strand reactions in the absence of Pol ε (lanes 1–4) or in the presence of 20 nM Pol ε (lanes 5–8). The reactions were 20 min …
Figure 4—figure supplement 1
Analysis of replication products in Pol α and Pol α−ε titrations illustrated in Figure 4A–C.
The autoradiographs shown in Figure 4 were analyzed by Typhoon laser scanner, and the lane profiles were normalized to the corresponding molecular weight at each pixel, as previously described (Kurth…
Figure 4—figure supplement 2
Pol ε excludes Pol α from the leading strand by taking over the primer whether RFC and PCNA are present or not.
(A) Autoradiogram of CMG mediated Pol α extension of the leading strand on the unprimed 3.2 kb forked DNA, in the presence or absence of Pol ε, as indicated in the figure. Reactions were performed …
Figure 4—figure supplement 3
CMG and Pol ε form a stable CMGE complex.
(A) Scheme of the bead assay to detect protein interactions. CMG-Pol ε complex was bound to Strep-Tactin magnetic beads via a strep-Pol ε, then washed and eluted with biotin; products were analyzed …
Figure 5 with 2 supplements
Pol δ functions on the ‘Pol ε suppressed’ lagging strand.
(A) Titration of Pol ε into lagging strand reactions containing Pol α/CMG results in inhibition of the lagging strand in the absence of RFC-PCNA. Similar reactions containing RFC-PCNA give even more …
Figure 5—figure supplement 1
Pol ε and RFC/PCNA inhibit Pol α DNA polymerase on the lagging strand, but Pol ε cannot extend primed sites with or without RFC/PCNA.
(A) Autoradiograph of lagging strand replication reactions using α32P-dGTP. CMG is pre-incubated and loaded on unprimed forked DNA, as described in ‘Materials and methods’, then either 10 nM Pol α …
Figure 5—figure supplement 2
RFC and PCNA inhibit Pol α DNA polymerase activity on ssDNA model templates.
Top: scheme of the assay. Primer extension assays utilized RPA-coated singly primed 5.4 kb φX174 ssDNA. RFC-PCNA is titrated into singly primed ssDNA replication assays containing 10 nM Pol α as …
Figure 6 with 1 supplement
Polymerases switch with Pol α on the leading strand.
(A) Alkaline agarose gel following the time course of leading strand extension using the indicated DNA polymerases. Reactions were assembled on unprimed forked DNA in presence of 24 nM CMG for 10 …
Figure 6—figure supplement 1
Pol δ does not inhibit the leading strand replication activity of Pol ε when all three polymerases are present.
(A) Autoradiographs of leading strand replication reactions using α32P-dCTP (left) or 32P-5′ labeled primed fork (right). CMG is pre-incubated and loaded on DNA, as described in ‘Materials and …
Figure 7
The leading and lagging strands are replicated at similar rates.
(A) Time course of leading–lagging strand replication reactions with all three polymerases at 10 nM each using either a pre-primed fork (Left panel) or unprimed fork (Right panel). Experiments were …
Figure 8
Exclusion reactions specify polymerase action at the eukaryotic fork.
(A) Pol α interacts with CMG to prime the leading and lagging strands. Pol α can extend DNA on both strands with CMG in vitro, but it lacks high fidelity and does not replicate bulk DNA in vivo. (B) …
