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. 2017 Mar 6;114(12):E2310–E2318. doi: 10.1073/pnas.1700280114

Fig. 1.

Fig. 1.

An unusual DNA polymerase from deep-sea vent phage NrS-1. (A) NrS-1 gene protein 28 shares weak homology to archaeal prim–pols. Alignment between NrS-1 gene protein 28 and three prim–pols encoded by archaeal plasmids (10) was made using CLC Sequence Viewer 6, and the regions containing homology are shown. Identical residues among archaeal prim–pols and those among prim–pols and NrS-1 gene protein 28 are highlighted by blue background. (B) SDS/PAGE gels showing purified proteins analyzed in this work. Lane 1, protein size marker; lane 2, NrS-1 polymerase; lane 3, N-terminal 400 residues of NrS-1 polymerase (N400); lane 4, C-terminal 318 residues of NrS-1 polymerase; lane 5, NrS-1 helicase; lane 6, NrS-1 ssDNA-binding protein; lane 7, NrS-1 ssDNA-binding protein (His-tag removed); lane 8, N-terminal 300 residues of NrS-1 polymerase (N300); lane 9, N-terminal 200 residues of NrS-1 polymerase (N200); lane 10, N300-D78A; lane 11, N300-D80A; lane 12, N300-D84A; lane 13, N300-E85A; lane 14, N300-H115A. All proteins carry an N-terminal His-tag except that in lane 7. (C) Extension of a primer by NrS-1 polymerase. A primed-template (40/100 nt) substrate in which the primer was 5′-32P–labeled and was incubated at 100 nM with 200 nM NrS-1 polymerase and 0.5 mM four dNTPs at 50 °C. Aliquots were removed at increasing time and analyzed on a 10% (wt/vol) TBE-urea gel (lanes 2–8). Lanes 1 and 9 contain 5′-32P–labeled 40 nt (primer) and the 100 nt complete complement to the template strand, respectively. (D) Nonspecific incorporation by NrS-1 polymerase. A primed-template (40/100 nt) substrate in which the primer was 5′-32P–labeled was incubated at 100 nM with 200 nM NrS-1 polymerase and 0.5 mM of various (d)NTPs at 50 °C for 30 min and then analyzed on a 10% (wt/vol) TBE-urea gel. Lane 1 is the 5′-32P–labeled 40 nt marker. Shown in red is the 5′ part of the DNA sequence to be synthesized downstream of the primer. (E) 100 nM each of the two 5′-32P–labeled 40-nt DNA strand (40 nt DNA-1 sequence 5′-TTTAGGTACCGGTGCCTAGCAGAAGGCCTAATTCTGCAAA-3′; 40 nt DNA-2 sequence 5′-TTTGCAGAATTAGGCCTTCTGCTAGGCACCGGTACCTAAA-3′) was incubated with 200 nM NrS-1 polymerase and 0.5 mM various (d)NTPs at 50 °C for 30 min and reaction products analyzed on a 10% (wt/vol) TBE-urea gel.