Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) sketch of the Polε holoenzyme proofreading process based on published studies in the absence of sliding clamp. The T/P unwinds by 3 bp and the unwound regions are kept separated during mismatch editing. The pol and exo sites are labeled. (B) Domain architectures of PolE1-4 and PCNA. Dashed lines indicate disordered regions. The structurally resolved regions (POLE1-NTD, i.e., Polε-core, and PCNA are colored by domains. POLE1-CTD and POLE2-4 are mobile relative to POLE1-NTD and invisible in EM maps. (C) The nucleotide sequence of the primer and template DNA. The mismatched primer 3’-end is highlighted in red. The T/P with a pre-existing mismatch cannot reach the exo site of Polε holoenzyme and binds Polε in a blocked state. (D) EM maps and atomic models of the Polε-PCNA–T/P complex in two blocked conformations. The maps and models are labeled and colored by domains as in (B). The distinct interactions of the Exo and thumb domains in the two conformations are highlighted by the red box.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques: Labeling, Sequencing

Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) SDS-PAGE gel of purified wild-type human Polε and PCNA. (B) Typical raw micrograph of the in vitro assembled complex. A total of 26,913 micrographs were recorded. (C) Selected 2D class averages in various views. (D) Workflow of cryo-EM data processing and 3D reconstruction in CryoSPARC (version 4.5.1), leading to the two 3D EM maps at 4.01 Å and 3.95 Å, respectively. (E) Angular distribution of particle images contributing to the two final 3D reconstructions. (F) Color-coded local resolution maps of the ternary complex in two blocked conformations. (G-H) Gold standard Fourier shell correlation (GSFSC) of the two EM maps (blue) and the model-to-EM map correlation curve (red) of the ternary complex in two blocked conformations.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques: SDS Page, Purification, In Vitro, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) SDS-PAGE gel of purified human Polε-core exo - and PCNA. (B) Typical raw micrograph out of the total 30,391 recorded movies. (C) Selected 2D class averages in various views. (D) Workflow of cryo-EM data processing and 3D reconstruction in CryoSPARC (version 4.5.1), leading to three 3D EM maps at 3.60 Å, 3.53 Å, and 3.11 Å average resolution, respectively. The final Bayesian polishing was performed by Relion-5.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques: SDS Page, Purification, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) Structure of the reported polymerization state (PDB ID 9B8T) of Polε–PCNA with a matched T/P. (B) Superimposition of the polymerization (gray) and mismatch-locking (color) states reveals that the Fingers domain transitions from the closed (gray) to open (dark green) conformation. (C) Superposition of the T/P reveals a 1-bp downward shift from the polymerization to the mismatch-locking state. (D) No major changes occur at the Polε-PCNA interface region from the polymerization (gray) to the mismatch-locking (color) states.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques:

Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) Superimposition on Polε-core of the mismatch-locking (color) and Pol-backtracking states (gray). Domain movements are indicated by red arrows. (B) A top view of the superimposition at the Polε-PCNA interface where most movements occur. The bulk of Polε-core is omitted for clarity. (C) Close-up view of changes in the mismatched region of T/P between the two states. The movements are indicated by red arrows and labeled. (D) Superimposition of T/P based on alignment on PCNA in both states. The DNA tilts 20° and moves with the thumb outwards by 9 Å. And the DNA moves down by 1 bp.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques: Labeling

Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) The mismatch-editing state structure colored by domains. (B) T/P DNA in sticks superimposed with the EM density in transparent surface view. The top orange box shows the clear DNA density around the exo - site of Polε, the bottom blue box shows that the weak DNA density inside the PCNA clamp. (C) Electrostatic surface of the exo - site with the primer 3′-end. (D) The Polε exo - site structure. The catalytic D275 and E277 coordinating two Mg 2+ in wild-type Polε are shown; they are mutated to A275 and A277 in Polε-exo - . (E) Close-up view of T/P around the exo - site. The T/P melts 6 bp then forms 4 out-of-register base pairs. The β-hairpin does not contact the template. Residues stabilizing the T/P are in sticks and labeled. (F) Sketch of T/P in editing state. (G) Comparison of the short Polɛ β-hairpin of (dark orange) and the longer RB69 gp43 β-hairpin (gray). The longer phage β-hairpin separates the template from primer.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques: Labeling, Comparison

Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) Comparison of the Pol-backtracking (gray) and mismatch-editing (color) states aligned on Polε-core. (B) Close-up view of the T/P and surrounding region. Red arrows indicate movements between the two states. Key residues are in sticks and labeled. (C) Structural changes near the Polε-PCNA interface. The top region of Polε-core is omitted for clarity. (D) Side-by-side comparison of the P-domain and T/P interaction in the two states. DNA-interacting residues are in sticks and labeled. (E) Comparison of the T/P in the two states reveal an overall translocation. The DNA tilts 10° toward the exo activity site. The duplex region spirals up by 6 bp, and 6 bp are unwound from the top primer 3′-end, enabling the primer to move 15 Å through exo channel into the exo site.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques: Comparison, Labeling, Translocation Assay, Activity Assay

Journal: bioRxiv

Article Title: The proofreading mechanism of the human leading strand DNA polymerase ε holoenzyme

doi: 10.1101/2025.04.11.648458

Figure Lengend Snippet: (A) Schematic representation of all five steps that are built based on experimental structures of the human Polε– PCNA holoenzyme. In step 1, Polε incorporates a mismatched base in the pol site and senses the mismatch via a Watson-Crick base-pairing checkpoint. This is modeled by Polε structure in the Pol state (PDB ID 9B8T). In step 2, Fingers domain flips up to an open position to arrest the pol activity, and the holoenzyme moves away from the mismatched 3′-end by 1 bp to prevent additional base incorporation. In step 3, the Polε linker helix rotates 10° and the thumb domain moves outwards by 9 Å. The movements put pressure on the 1-bp backtracked T/P. In step 4, the P-domain tilts 12° against the PCNA, and the linker helix rotates 36°, causing the holoenzyme to backtrack by 6 bp and unwind 6 bp from the primer 3′-end. The 6 unwound bp rebind and form 4 out-of-register bp and to insert the mismatched primer 3′-end into the exo site. In step 5, the primer 3′ mismatch is excised by the exo activity. Next (not shown), in an expected reverse process not yet well characterized, the holoenzyme returns the T/P to the pol site and resumes DNA synthesis. (B) Schematic describing the mismatch DNA translocation during the Polε–PCNA holoenzyme proofreading process. The horizontal dashed line indicates the post insertion site of Polε. The movement of DNA between each step is indicated by red arrows.

Article Snippet: The cDNAs of human Polε (POLE1-4) were acquired from DNASU Plasmid Repository, and the cDNA of human PCNA was obtained from Addgene.

Techniques: Activity Assay, DNA Synthesis, Translocation Assay

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Antibodies used for Western blots.

Article Snippet: Mouse anti-human PCNA , Wuhan Boster Bio, China , BM0104 , 1:1000.

Techniques: Western Blot

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Effects of BFA and TM on the expression of PCNA in HepG2 cells. HepG2 cells were treated with BFA (0.25 mg/L) and TM (1 mg/L), either individually or in combination, for 24 h. The protein levels of PCNA were determined using Western blot assay. Data are presented as means ± SD from three independent experiments performed in triplicate. ∗ P < 0.05 vs. control group; # P < 0.05 vs. DMSO (0.1% v/v, vehicle control) group. Abbreviations: BFA, brefeldin A; DMSO, dimethyl sulfoxide; PCNA, proliferating cell nuclear antigen; SD, standard deviation; TM, tunicamycin.

Article Snippet: Mouse anti-human PCNA , Wuhan Boster Bio, China , BM0104 , 1:1000.

Techniques: Expressing, Western Blot, Control, Standard Deviation

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Antibodies used for Western blots.

Article Snippet: Mouse anti-human PCNA , Wuhan Boster Bio, China , BM0104 , 1:1000.

Techniques: Western Blot