A major clinical problem in the use of cisplatin to treat cancers is tumor resistance1,3
. The ability of human Polη to bypass cisplatin-DNA adducts formed during cancer chemotherapy is one important mechanism by which tumors appear to gain such resistance4
. We show here that human Polη can accommodate a bulky PtGpG DNA crosslink within its active site cleft without any major rearrangement of the enzyme. The specificity of human Polη for PtGpG, as compared to other DNA polymerases, derives from an active site cleft that is sufficiently open to permit not only near perfect Watson-Crick geometry of the nascent PtGpG
•dCTP base pair but also to accommodate the steeply inclined 5′G of PtGpG without any steric hindrance. The specificity for PtGpG is further augmented by residues Gln38 and Ser62 that interact with the 3′G and 5′G of the PtGpG adduct, respectively, and Arg61 that interacts with incoming dCTP. Amongst human TLS polymerases5
, Arg61 and Ser62 are unique to Polη, while Gln38 occurs only in Polη and Polɭ. In the structure of yeast Polη in complex with a cisplatin-DNA adduct, Gln55 and Arg73 make similar local interactions with DNA as Gln38 and Arg61 in human Polη. By contrast, in the structure of prokaryotic Dpo4–PtGpG complex that lacks these amino acids, the 3′G is tilted and shifted towards the major groove and unable to make a WC base pair with incoming dCTP18
. Thus, interactions with Gln38 and Ser62 appear to be necessary to keep the PtGpG optimally oriented for proper WC base pairing between 3′G and incoming dCTP.
The human Polη active site cleft is well-poised for catalysis and for incorporation of C opposite the 3′G of PtGpG. Thus, even though PtGpG is a larger and more distorted intrastrand cross-link than a cis-syn
T-T dimer, it does not lead to any significant perturbation of the human Polη active site or impact the ability of the polymerase to carry out catalysis. The primer terminus is well-aligned for incorporation of C opposite the 3′G of PtGpG adduct. By contrast, based on our biochemical studies, human Polη is markedly inhibited in extending from the 3′G, 5′G, or the next templating residue of the PtGpG adduct (Supplementary Fig. 3
). This may be one reason why we were unsuccessful in obtaining suitable crystals of a ternary complex of human Polη inserting a nucleotide opposite the 5′G of the PtGpG adduct. A recurring theme in TLS is that lesion bypass often requires the sequential action of two polymerases, an “inserter” and an “extender”5
The inserter is efficient at insertion of an incoming nucleotide across from the lesion and the extender is recruited to add bases downstream of the lesion. In eukaryotes, Polζ, a B-family polymerase, is specialized for the extension step of lesion bypass19
, and it is conceivable that Polζ (or another polymerase) is recruited in human cells to complete PtGpG bypass.
The ability of human Polη to replicate across from a cisplatin adduct makes it an attractive target for cancer therapy. A pressing question is whether one can design or identify inhibitors that are specific for human Polη ? The fact that the shape of the human Polη active site cleft is different from that of other DNA polymerases suggest that it would be possible to derive small molecules that can specifically inhibit human Polη. The human Polη active site cleft, for example, is more open than in other DNA polymerases and it lacks elements such as the N-clasp in human Polκ and the N-digit in human Rev120,21
. At the same time, the presence of unique (Arg61 and Ser62) and nearly unique (Gln38) residues within the active site cleft offer the possibility of specific hydrogen bonds with unique small molecules.
Preparation of protein and DNA for crystallization
The catalytic core of human Polη (residues 1–432) harboring an N-terminal hexa-histadine (6XHis) tag and a C406M mutation was overexpressed in Escherichia coli
and purified as previously described by Biertumpfel et al8
. Briefly, the 6XHis tag was removed via overnight incubation with PreScission protease and the protein subsequently purified via ion-exchange (MonoS) and size-exclusion (Superdex75) chromatography, prior to concentration of the protein for crystallization experiments. A HPLC purified 13–nt template strand (5′ – CTTGG
TCTCCTCC – 3′) was prepared (Integrated DNA Technologies Inc.) and a cis
-Diamminedichloroplatinum(II) (cisplatin; GFS Chemicals Inc.) intrastrand d(GpG) cross-link introduced between the two adjacent guanines based on the method described by Gelasco and Lippard16
. The cisplatin modified template strand was further purified by HPLC on a reversed-phase C18 column (Waters Inc.) A 9–nt primer strand harboring a dideoxyadenosine at its 3′ end (5′ –TGGAGGAGAdd
- 3′) was annealed in the molar ratio of 1:1 to the 13–nt cisplatin-modified template strand to yield the 9–nt/13–nt primer/template.
Preparation of proteins for biochemical analysis
The full length wild-type and the various mutant versions of human Polη were expressed in yeast Saccharomyces cerevisiae
and purified by glutathione-Sepharose affinity chromatography as described previously22
, except that proteins contained tandem N-terminal GST and Flag tags. The GST tags were removed by treatment with PreScission protease, leaving an N-terminal Flag tag attached to each protein. Proteins were quantified by Coomassie stained SDS-PAGE analysis. Mutations were introduced into the wild type open reading frame by PCR using oligonucleotides containing site specific mutations and were confirmed by sequencing prior to expression.
DNA polymerase assays
DNA substrates consisted of a radiolabeled oligonucleotide primer annealed to a 78-nt oligonucleotide DNA template by heating a mixture of primer/template at a 1:1.5 molar ratio to 95°C and allowing it to cool to room temperature for several hours. The template 78-mer oligonucleotide contained the sequence 5′AGCAAGTCAC CAATGTCTAA GAGTTTCTTGGTCTCCTCCT ACACTGGAGT ACCGGAGCAT CGTCGTGACT GGGAAAAC-3′ and was either undamaged or harbored a G-Pt-G intrastrand cross-link at the underlined position. The sequence of the running start primer is 5′CGACGATGCTCCGGTACTCCAGTGTAG 3′. For steady-state kinetic analyses of nucleotide insertion opposite the 3′G of either the undamaged GG or GG cisplatin template, the primer 5′GATGCTCCGG TACTCCAGTG TAGGAGGAGA 3′ was used. The standard DNA polymerase reaction (5 μl) contained 25 mM Tris·HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiolthreitol, 100 μg ml−1 BSA, 10% glycerol, and 10 nM DNA substrate. For running start assays, 25 μM each of dATP, dTTP, dCTP, and dGTP (Roche Biochemicals) was included. For steady state kinetic experiments, only dCTP was included at concentrations ranging from 0.05 to 50 μM. Reactions containing wild type and mutant Polη (0.05 nM to 0.5 nM) were carried out at 37 °C for 5-10 min. Reactions were terminated by the addition of 6 volumes of loading buffer (95% formamide, 0.05% cyanol blue, and 0.05% bromophenol blue) before resolving on 12% polyacrylamide gels containing 8M urea. Gels were dried before autoradiography.
Steady-state kinetic analysis
Steady-state kinetic analyses for deoxynucleotide incorporation were performed as described22
. Gel band intensities of the substrate and products of the deoxynucleotide incorporation reactions were quantified by using a PhosphorImager and the IMAGEQUANT software (Molecular Dynamics). The observed rate of deoxynucleotide incorporation, vobs
was determined by dividing the amount of product formed by the reaction time and protein concentration. The vobs
was graphed as a function of the deoxynucleotide concentration, and the data were fit to the Michaelis-Menten equation describing a hyperbola: vobs
[E] × [dNTP])/(Km
+ [dNTP]). From the best fit curve, the apparent Km
steady-state kinetics parameter were obtained for the incorporation of dCTP by the wild type and mutant Polη and the efficiencies of nucleotide incorporation (kcat
The cisplatin (PtGpG) ternary complex was prepared by mixing human Polη with the cisplatin modified primer/template in a 1:1.1 molar ratio in the presence of 2mM dCTP and 5mM MgCl2. This mix was incubated for at least an hour before crystallization. Thin needle-like crystals were obtained in 0.1M Bis-Tris (pH 5.5) and 30%-35% (w/v) of PEG 1500 by the vapor diffusion method at 20°C. Several rounds of microseeding (15-30% PEG 1500) were required to produce good quality crystals. For data collection, the crystals were cryoprotected by step-wise soaks for 5 min in mother liquor solutions containing 5-30% (v/v) of glycerol, and then flash frozen in liquid nitrogen. X-ray diffraction data were collected on beamline X25 at Brookhaven National Laboratory (BNL). The crystals diffract to 2.32 Å resolution with synchrotron radiation at the platinum LIII absorption edge (λ = 1.0722 Å). Crystals belong to space group P6(1) with unit cell dimensions a = 98.5 Å, b = 98.5 Å and c = 82.6 Å. Matthew’s coefficient calculation indicates one protein-DNA complex in the asymmetric unit.
Structure determination and refinement
The structure was solved by molecular replacement (MR), using the human Polη ternary structure (PDB 3MR2), with the DNA, incoming nucleotide, metals, and water molecules omitted, as the starting model8
. The program PHASER gave a unique MR solution23
. The first round of refinement and map calculation was carried out with just the enzyme using REFMAC24
. The electron density maps (2Fo-Fc and Fo-Fc) showed unambiguous densities for the cisplatin 1, 2 intrastrand crosslink between the adjacent guanines in the template strand (), primer strand, incoming nucleotide and metal, which were then iteratively built into the map using COOT25
. Iterative rounds of refinement and water picking were performed with REFMAC, and model building with COOT. The final model has excellent stereochemistry (), as shown by Molprobity with 97.9% of the residues in the most favored regions of the Ramachandran plot17
. Figures were prepared using PyMol (http://www.pymol.org