primer extension  (New England Biolabs)

Journal: Nature Communications

Article Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase

doi: 10.1038/s41467-023-39441-1

Figure Lengend Snippet: a Domain architecture of the four subunits of the holoenzyme. The red asterisk indicates the polymerase activity in the catalytic NTD of Pol1 which is referred to as Pol1-core. The yellow asterisk indicates the primase activity in Pri1. b A sketch of Pol α RNA primer and DNA primer synthesis steps. c – g Six cryo-EM maps of Pol α in different states of catalysis: Pol α in the absence of T/P exists in the Apo state (conformers I and II), c ), Pol α–T (T refers to template DNA) is in the primer initiation state ( d ), Pol α–T/P8 (T/P8 refers to the 8-nt RNA primer annealed to the template) is in the RNA synthesis state ( e ), Pol α–T/P10 (T/P10 refers to the 10-nt RNA primer annealed to the template) is in the post RNA hand-off state ( f ), and Pol α–T/P15 (T/P15 refers to the 10-nt RNA and 5-nt DNA chimeric 15-mer primer annealed to the template) is in the DNA elongation state.

Article Snippet: To capture the transition (RNA hand-off state from Pri1 to Pol1), Pol α was individually incubated with the 9-nt, 10-nt, and 11-nt RNA primers annealed with the 60-nt DNA template (T/P9, T/P10, and T/P11) at 30 °C for 5 min. To induce the DNA primer extension state, we incubated the enzyme with the 15-nt RNA-DNA chimeric primer annealed to the 60-nt DNA template (T/P15) using the same conditions as described above before making cryo-EM grids.

Techniques: Activity Assay, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase

doi: 10.1038/s41467-023-39441-1

Figure Lengend Snippet: a Front cartoon view of Pol α–T/P10 in the post RNA hand-off state. Insert at lower left is a typical 2D class average. The lower right panel shows an enlarged view of the interface between Pol1-core and RNA/DNA. The thumb and palm subdomains bind the RNA primer nt 2–9, and the thumb is flush with P5. b Comparison of Pol1-core in the RNA synthesis state (Pol α–T/P8, gray) and primer hand-off state (Pol α-T/P10, color). Pol1-core rotates 60° to bind the RNA/DNA duplex. Lower left panel shows the change of Pol1-core in the post RNA hand-off state will occupy the space of Pri1. Lower right panel shows the N-term of Pol1-core may interact with Pri1-NTD as seen in Apo conf II. c Changes from post RNA hand-off state to DNA elongation state. Pol1-core captures the RNA/DNA while Pri1 dissociates from template DNA. d Cartoon model showing RNA primer hand-off from the primase (Pri1) to the polymerase (Pol1-core).

Article Snippet: To capture the transition (RNA hand-off state from Pri1 to Pol1), Pol α was individually incubated with the 9-nt, 10-nt, and 11-nt RNA primers annealed with the 60-nt DNA template (T/P9, T/P10, and T/P11) at 30 °C for 5 min. To induce the DNA primer extension state, we incubated the enzyme with the 15-nt RNA-DNA chimeric primer annealed to the 60-nt DNA template (T/P15) using the same conditions as described above before making cryo-EM grids.

Techniques:

Journal: Nature Communications

Article Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase

doi: 10.1038/s41467-023-39441-1

Figure Lengend Snippet: a Front cartoon view of Pol α–T/P8. Inserted at lower left is a typical 2D class average showing the domain arrangement and the partially flexible Pol1-core. b Top view of Pol α–T/P8 with Pol12 and Pol1-CTD removed for clarity. c Comparison on how Pri1 and Pri2-CTD interact with template and primer in the apo state conf II (1st row), primer initiation state (2nd and 3rd rows), and RNA priming state (4th row). The human Pol α-primase in the 3rd row is from the PDB ( 8D0K ). These structures are shown in a side (left column) and a top (middle column) view. The right column is a cartoon model showing how Pri1 and Pri2-CTD are arranged to load template DNA and synthesize an RNA primer.

Article Snippet: To capture the transition (RNA hand-off state from Pri1 to Pol1), Pol α was individually incubated with the 9-nt, 10-nt, and 11-nt RNA primers annealed with the 60-nt DNA template (T/P9, T/P10, and T/P11) at 30 °C for 5 min. To induce the DNA primer extension state, we incubated the enzyme with the 15-nt RNA-DNA chimeric primer annealed to the 60-nt DNA template (T/P15) using the same conditions as described above before making cryo-EM grids.

Techniques:

Journal: Nature Communications

Article Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase

doi: 10.1038/s41467-023-39441-1

Figure Lengend Snippet: a Front cartoon view of Pol α–T/P15. Three insets show detailed contacts between Pol1-core and T/P. Inserted at lower left is a typical 2D class average showing the largely flexible Pri1. b Schematic diagram of the interactions of Pol α’s thumb and palm subdomains with the T/P. c Comparison of RNA priming state, post-RNA hand-off state, and DNA elongation state. The structures are aligned based on Pri2-CTD. The Pol1-core thumb touches the bottom of the RNA/DNA duplex in the RNA synthesis state. The Pol1-core thumb and palm subdomains fit the first minor groove and interact with the RNA primer (P1–P10 region) in the post-RNA hand-off state. In the DNA elongation state, the thumb and palm subdomains of Pol1-core fit the second minor groove and interact with primer strands nt 1–2 and 9–14, as well as the template strand nt 9–14. d Sketch of DNA polymerase loading and DNA primer elongation. The schematic DNA drawings were modified from a DNA template ( https://www.vecteezy.com/vector-art/3069633-dna-molecule-icon-vector-illustration-on-white-background ).

Article Snippet: To capture the transition (RNA hand-off state from Pri1 to Pol1), Pol α was individually incubated with the 9-nt, 10-nt, and 11-nt RNA primers annealed with the 60-nt DNA template (T/P9, T/P10, and T/P11) at 30 °C for 5 min. To induce the DNA primer extension state, we incubated the enzyme with the 15-nt RNA-DNA chimeric primer annealed to the 60-nt DNA template (T/P15) using the same conditions as described above before making cryo-EM grids.

Techniques: Modification, Plasmid Preparation

Journal: Nature Communications

Article Title: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase

doi: 10.1038/s41467-023-39441-1

Figure Lengend Snippet: 1 Pol α fluctuates between apo conformers I and II in the absence of a DNA template, with the Pol1-core catalytic site being blocked by the Pol1-CTD/Pol12 platform and Pri2-CTD and Pri1 moving about. 2 In the primer initiation state, Pol α engages a template DNA. Pol1-core dissociates from the platform. Pri2-CTD directs the template DNA to the primase active site in Pri1. 3 In the RNA synthesis state, Pri1 synthesizes the RNA primer on the DNA template. The RNA/DNA hybrid duplex displaces Pri2-CTD from Pri1, allowing Pol1-core to engage the growing T/P. 4 In the RNA primer hand-off state, Pol1-core captures the 3’-end of the RNA primer and expels Pri1 from template DNA. This completes the RNA primer hand-off from Pri1 to Pol1-core. 5, 6 In the DNA elongation state, Pol1-core spirals around the RNA/DNA duplex to synthesize the DNA primer. The schematic DNA drawings were modified from a DNA template ( https://www.vecteezy.com/vector-art/3069633-dna-molecule-icon-vector-illustration-on-white-background ).

Article Snippet: To capture the transition (RNA hand-off state from Pri1 to Pol1), Pol α was individually incubated with the 9-nt, 10-nt, and 11-nt RNA primers annealed with the 60-nt DNA template (T/P9, T/P10, and T/P11) at 30 °C for 5 min. To induce the DNA primer extension state, we incubated the enzyme with the 15-nt RNA-DNA chimeric primer annealed to the 60-nt DNA template (T/P15) using the same conditions as described above before making cryo-EM grids.

Techniques: Modification, Plasmid Preparation