Despite differences in sequence, structure, and function, all DNA polymerases follow essentially the same reaction pathway 1; 2; 3; 4; 5; 17; 18
. That is, preceding phosphoryl transfer, DNA polymerases first bind the template-primer to form the preinsertion polymerase-DNA binary complex, and then bind incoming dNTP to form the preinsertion polymerase-DNA-dNTP ternary complex. This is then followed by a step leading to the formation of a phosphodiester bond between the α-phosphate of dNTP and the 3′OH at the primer terminus. Together, these transitions preceding phosphoryl transfer serve as kinetic checkpoints for the rejection of incorrect nucleotides early in the reaction pathway 2
. From pre-steady state kinetic studies it has been inferred that classical polymerases as well as Polη undergo a rate-limiting conformational change step before the phosphoryl transfer reaction 2; 3; 11
. In the case of classical polymerases, this rate-limiting step was considered to be the large “open-to-closed” conformational transition in the fingers domain observed in crystal structures, which brings the domain closer to the active site residues 1; 2; 3
. However, from recent kinetic studies, the motion of the fingers appears to be relatively fast 19; 20; 21; 22; 23
, and it has been suggested that the basis of the rate limiting step in the catalytic cycles of these classical polymerases may correspond to a change in metal ion occupancy in the active site 24; 25; 26
. A similar mechanism may operate in Polη. From a comparison of the binary and ternary hPolη stuctures presented here, the fingers domain occupies almost the same position with respect to the palm domain (and the active site residues) in the presence or absence of dNTP. The residues that normally interact with the triphosphate moiety of dNTP (Tyr52, Arg55 and Lys231) and the active site residues Asp13, Asp115, and Glu116) are similarly disposed in the binary and ternary complexes, suggesting that Polη is, for the most part, pre-configured for dNTP binding and catalysis upon binding the template-primer. A notable difference between the structures is the absence of catalytic metals in the binary complex. Analogous to the more classical polymerases, we suggest that the rate- limiting conformational change step in Polη may correspond to the rate-limiting entry of catalytic metals, Mg2+
A and Mg2+
B, in the active site.
The mechanism we propose here for Polη differs from that suggested previously 15
. Based on the observation of more than one DNA orientation in the ternary structures of yPolη with cisplatin adducts, it has been suggested, for example, that the DNA “rotates” from a nonproductive to a productive configuration on dNTP binding 15
. However, there is no indication from our comparison of the hPolη binary and ternary structures that the DNA binds in more than one configuration, with the PAD maintaining a tight and almost identical grip on the DNA in both complexes. Interestingly, when Dpo4, an archaeal homolog of Polκ, binds 8-oxoG modified template-primer, the PAD slides or translocates by one nucleotide step in the presence of dNTP, while the thumb domain-DNA contacts remain fixed 27
. By contrast, the hPolη PAD maintains the same register with respect to template-primer and other domains in the absence or presence of dNTP. Overall, the hPolη structures are more reminiscent of binary and ternary of complexes human Polι 28
, wherein the catalytic pocket is essentially preformed and the binding of dNTP imposes a local rather than any large conformational change in the inter-domain positions.
In summary, we show here that the hPolη domains move only slightly on dNTP binding and that the polymerase by and large is pre-aligned for dNTP binding and catalysis. We also show that there is no major reorientation of the DNA from a nonproductive to a productive configuration, as previously suggested15
. Instead, as with the classical polymerases, we suggest that the rate- limiting conformational change step in the Polη replication cycle likely corresponds to a rate-limiting entry of catalytic metals in the active site. A similar conclusion was reached for Dpo4 based on structures with matched or mismatched incoming dNTP 29
, suggesting that metal ion coordination may be the key rate-limiting step for most DNA polymerases.