Journal: Nature Communications

Article Title: Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis

doi: 10.1038/s41467-025-61245-8

Figure Lengend Snippet: ( A ) Scheme of the time-lapse X-ray crystallography approach. ( B ) Overall structure of the pol ι-DNA binary substrate complex. ( C ) Close-up of the active site of the pol ι-DNA binary substrate complex with the template base labeled n and the primer-terminal base pair labeled n-1. ( D ) Close-up of the active site of the pol ι-DNA-dTTP substrate complex with the nascent base pair labeled n , the primer-terminal base pair labeled n -1, and the base on the 5′ side of the template base labeled n + 1 and with a polder map contoured at σ = 3.0 around the two active site calcium ions and the incoming dTTP (green mesh). The conclusion that the electron density shown here corresponds to calcium ions rather than water molecules or sodium ions is supported by observing an increase in R-free when modeling water molecules or sodium ions. ( E ) Close-up of the active site of the pol ι-DNA-dTTP substrate complex showing the polar contacts between the dTTP, the Ca 2+ ions, and select amino acid residues.

Article Snippet: Pol ι (20 mg/ml), the DNA substrate (500 μM), and dTTP (1 mM, New England Biolabs, catalog number N0446S) were co-crystallized in the presence of 5 mM CaCl 2 .

Techniques: Labeling

Journal: Nature Communications

Article Title: Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis

doi: 10.1038/s41467-025-61245-8

Figure Lengend Snippet: ( A ) Close-up of the active site of the pol ι complex with pyrophosphate bound and with the newly formed base pair labeled n, the primer-terminal base pair labeled n-1, and the base on the 5′ side of the template base labeled n + 1 and with a polder map contoured at σ = 3.0 around the pyrophosphate (green mesh). ( B ) Overlay of the DNA and dTTP substrates of the pol ι-DNA-dTTP ternary complex (white) and the DNA of the pol ι complex with pyrophosphate bound (yellow). The distances moved by the α-phosphate of the dTTP, the 3′ oxygen of the primer terminus, and the two metal ions is indicated. ( C ) Close-up of the active sites of the pol ι complex with two monophosphate products bound and with the newly formed base pair labeled n, the primer-terminal base pair labeled n -1, and the base on the 5′ side of the template base labeled n + 1 and with a polder map contoured at σ = 3.0 around the two monophosphates (green mesh). The conclusion that the electron density shown here corresponds to monophosphates rather than water molecules is supported by observing an increase in R-free when modeling water molecules. ( D ) Graph showing the production of monophosphate by pol ι in the presence of DNA and dTTP. Data are presented as mean values ± SEM. Four technical replicates of each experimental condition were carried out. Source data are provided as a Source Data file. The p values were derived from one-way ANOVA using GraphPad Prism, and **** indicates p < 0.0001.

Article Snippet: Pol ι (20 mg/ml), the DNA substrate (500 μM), and dTTP (1 mM, New England Biolabs, catalog number N0446S) were co-crystallized in the presence of 5 mM CaCl 2 .

Techniques: Labeling, Derivative Assay

Journal: Nature Communications

Article Title: Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis

doi: 10.1038/s41467-025-61245-8

Figure Lengend Snippet: ( A ) Nucleotide-incorporation activity of the wild-type, mutant Y61A, and mutant R71A pol ι proteins. These experiments were repeated four times to ensure reproducibility. The uncropped gel images are provided as a Source Data file. ( B ) Close-up of the active site of the mutant R71A substrate complex with the newly formed base pair labeled n, the primer-terminal base pair labeled n -1, and the base on the 5′ side of the template base labeled n + 1 and with a polder map contoured at σ = 3.0 showing the weak electron density corresponding to the incoming dTTP (green mesh).

Article Snippet: Pol ι (20 mg/ml), the DNA substrate (500 μM), and dTTP (1 mM, New England Biolabs, catalog number N0446S) were co-crystallized in the presence of 5 mM CaCl 2 .

Techniques: Activity Assay, Mutagenesis, Labeling

Journal: Nature Communications

Article Title: Visualizing DNA polymerase ι catalyze Hoogsteen-directed DNA synthesis

doi: 10.1038/s41467-025-61245-8

Figure Lengend Snippet: ( A ) Close-up of the active site of the pol ι-DNA-dTTP substrate complex with an overlay of 20 structures of the nascent base pair (shown as sticks) obtained after every 50 ns of simulation time for the first replicate. ( B ) Graph showing the glycosidic angle of the template nucleotide as a function of time. The shaded region (0 to 90°) represents the typical range for a Hoogsteen base pair. ( C , D ) Same as above for the pol ι complex bound to the pyrophosphate product. ( E , F ) Same as above for the pol ι complex bound to two monophosphate products. ( G , H ) Same as above for the pol ι complex bound to one monophosphate product. ( I , J ) Same as above for the pol ι binary product complex.

Article Snippet: Pol ι (20 mg/ml), the DNA substrate (500 μM), and dTTP (1 mM, New England Biolabs, catalog number N0446S) were co-crystallized in the presence of 5 mM CaCl 2 .

Techniques: