Site-specific urea lesion-containing
12-mer template oligodeoxynucleotides corresponding to each urea-containing
strand were annealed with an equimolar amount of 8-mer oligodeoxynucleotide
primers (Chart 2 ) in
100 mM NaCl, 50 μM EDTA (sodium salt), and 20 mM Tris–HCl
(pH 8.5) at room temperature. They were kept on ice for 30 min. To
first make the binary complex of hPol η and urea-containing
DNA template primer, protein in 450 mM KCl, 5% glycerol, 1 mM TCEP,
3 mM DTT, and 20 mM Tris–HCl (pH 7.5), and DNA were mixed in
a 1:1.1 molar ratio, followed by incubation at room temperature for
10 min. To reduce the salt concentration to 150 mM KCl, dilution buffer
containing a concentration of 5 mM MgCl, 5% glycerol, 1 mM TCEP, 3
mM DTT, and 20 mM Tris–HCl (pH 7.5) was added. The samples
were concentrated using 10 kDa cutoff amicon centrifugal filters (Millipore
Sigma, Burlington, MA) to yield 2 mg/mL protein concentration. To
assemble ternary complexes, dNTP analogs 2′-deoxyadenosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dAMPnPP), 2′-deoxycytidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dCMPnPP), 2′-deoxyguanosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dGMPnPP), and 2′-deoxythymidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dTMPnPP) (Jena BioScience, Jena, Germany) were added
separately to a 10 mM final concentration into a tube that contained
concentrated hPol η-DNA binary complex. Solutions were kept
on ice for 30 min before setting up 24-well crystallization plates.
For each ternary complex crystallization, we used the hanging drop
vapor diffusion method in combination with a 700 mL reservoir containing
polyethylene glycol monomethyl ether 2000 (PEG MME2000) (Hampton Research,
Aliso Viejo, CA) (14–24%), 5 mM MgCl2, and 0.1 M
MES hydrate (Millipore Sigma, Burlington, MA) at one of three pH values
of pH 5.6, pH 6.0, and pH 6.5.18 (link),30 (link) For each drop, 0.8
μL of ternary complex was mixed with 0.8 μL of a reservoir
solution. Plates were incubated either at 18 °C or at room temperature.
Crystals were observed for samples corresponding to both urea-containing
DNA strands and all four dNTPs at different pH and PEG concentrations
in 1 to 2 days. Diffraction-quality crystals were obtained after incubating
plates for 1 week.
12-mer template oligodeoxynucleotides corresponding to each urea-containing
strand were annealed with an equimolar amount of 8-mer oligodeoxynucleotide
primers (
100 mM NaCl, 50 μM EDTA (sodium salt), and 20 mM Tris–HCl
(pH 8.5) at room temperature. They were kept on ice for 30 min. To
first make the binary complex of hPol η and urea-containing
DNA template primer, protein in 450 mM KCl, 5% glycerol, 1 mM TCEP,
3 mM DTT, and 20 mM Tris–HCl (pH 7.5), and DNA were mixed in
a 1:1.1 molar ratio, followed by incubation at room temperature for
10 min. To reduce the salt concentration to 150 mM KCl, dilution buffer
containing a concentration of 5 mM MgCl, 5% glycerol, 1 mM TCEP, 3
mM DTT, and 20 mM Tris–HCl (pH 7.5) was added. The samples
were concentrated using 10 kDa cutoff amicon centrifugal filters (Millipore
Sigma, Burlington, MA) to yield 2 mg/mL protein concentration. To
assemble ternary complexes, dNTP analogs 2′-deoxyadenosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dAMPnPP), 2′-deoxycytidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dCMPnPP), 2′-deoxyguanosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dGMPnPP), and 2′-deoxythymidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dTMPnPP) (Jena BioScience, Jena, Germany) were added
separately to a 10 mM final concentration into a tube that contained
concentrated hPol η-DNA binary complex. Solutions were kept
on ice for 30 min before setting up 24-well crystallization plates.
For each ternary complex crystallization, we used the hanging drop
vapor diffusion method in combination with a 700 mL reservoir containing
polyethylene glycol monomethyl ether 2000 (PEG MME2000) (Hampton Research,
Aliso Viejo, CA) (14–24%), 5 mM MgCl2, and 0.1 M
MES hydrate (Millipore Sigma, Burlington, MA) at one of three pH values
of pH 5.6, pH 6.0, and pH 6.5.18 (link),30 (link) For each drop, 0.8
μL of ternary complex was mixed with 0.8 μL of a reservoir
solution. Plates were incubated either at 18 °C or at room temperature.
Crystals were observed for samples corresponding to both urea-containing
DNA strands and all four dNTPs at different pH and PEG concentrations
in 1 to 2 days. Diffraction-quality crystals were obtained after incubating
plates for 1 week.
