Structure of a gap-filling intermediate
The first crystal structure of pol λ captured the polymerase in an inactive binary complex in which the enzyme was bound to a two-nucleotide gap, but without a dNTP present19
. In this structure, the 8 kDa domain was bound to the 5'-phosphate end of the gap, but the polymerase active site was not productively engaged at the 3' end of the gap. Although not representing a catalytically competent gap filling intermediate, this structure suggested that, in the absence of an incoming nucleotide, the polymerase might preferentially bind to the 5'-end of a gap, a feature that appeared consistent with a role in gap filling.
In order for the first step of gap-filling to occur, the active site of the polymerase must be bound in its catalytic conformation at the 3'-end of the primer. If, as suggested by the biochemical data, polymerization occurs while the 8kDa domain is engaging the 5'-phosphate, it is necessary to invoke a conformational change leading to an intermediate where, either through DNA or protein rearrangements, the polymerase active site would bind the 3' DNA end in a catalytic conformation while the 8 kDa domain would remain bound to the 5'-end of the gap. Because such a conformation is not seen in the 2-nucleotide gap binary complex (1RZT), we hypothesized that it could be dependent on dNTP binding. Thus, we attempted to determine the structure of a ternary complex of human pol λ bound to a 2-nucleotide gap with a correct dNTP bound in a pre-catalytic state. We grew crystals of pol λ after mixing the enzyme with a three-nucleotide gap in the presence of ddTTP, with the expectation that incorporation of ddTMP would result in a dideoxy-terminated two-nucleotide gap with ddTTP bound in the polymerase active site (see Methods). We obtained crystals that diffracted to a resolution of 1.95Å () and we were able to solve the structure by molecular replacement (see Methods). The structure () shows pol λ bound to DNA while attempting to incorporate a correct nucleotide opposite the first of the two single-stranded nucleotides in the gap. The polymerase binds in a remarkably similar manner to what was observed in previous ternary complex structures involving a single nucleotide gap11
, despite the fact that the gap is one nucleotide longer (rmsd of 0.935 Å for 316 C-a atoms). All the polymerase active site residues are in conformations indistinguishable from those observed in a single-nucleotide gap structure, indicating that this structure represents a functional complex. Moreover, the 8 kDa domain is engaged in binding the 5'-phosphate, and does so in a similar conformation as is observed when the enzyme is in complex with a 1-nucleotide gap. The solution to this apparent paradox is that the template strand is scrunched, where one of the template nucleotides in the 2-nucleotide gap is extrahelical (), and bound in a manner that results in very little distortion of helix geometry (). The largest difference is in the position of the phosphate 5'- to the extrahelical base (). Importantly, the 3' primer terminus and the 5' phosphate of the downstream primer (arrows in ) remain in the same relative positions, and only a slight difference is observed in the conformation of the downstream DNA and the 8 kDa. By adopting this conformation, Pol λ engages both sides of the gap in the absence of functionally relevant protein conformational changes and without changing the relative locations of the polymerase and 8 kDa domains. For the latter reason, we refer to this as “scrunching”, by analogy to what was previously observed with RNA polymerases20,21,22
Data collection and refinement statistics
Figure 1 Structure of Pol λ bound to a two-nucleotide gap. a. Overview of the structure. The DNA polymerase was crystallized in a pre-catalytic state, prior to incorporation of the incoming dTTP (magenta). The primer (P) and downstream primer (D) strands (more ...)
A binding pocket for purines and pyrimidines
Inspection of the structure reveals that the extrahelical nucleotide is bound in a pocket formed by the side chains of three amino acids (). When the nucleotide bound in the pocket is an adenine, Leu277 and His511 provide hydrophobic contacts with the extrahelical base, while Arg514 mainly interacts with the phosphate 3' to that nucleotide. To determine if this pocket also accommodates pyrimidines, we crystallized pol λ in complex with a 2-nucleotide gap and incoming dTTP as before, but altered the template sequence so that dCMP is present as the second single-stranded nucleotide in the gap. These crystals diffracted to a resolution of 1.95Å (see and Methods), and the resulting electron density indicates cytosine is indeed bound in the pocket (). Binding is similar to that observed with adenine (), except that the side chain of Arg514 adopts a different conformation that allows it to hydrogen bond with the O2 atom in the cytosine base, while Leu277 appears to undergo a slight rearrangement to maximize contact with the smaller pyrimidine base. These results suggest that the three residues comprising the binding pocket for the extrahelical base share an analogous role for all four bases normally present in DNA, and further suggest that the pocket may have a role in stabilizing the scrunched intermediate. The functional significance of this pocket is further implied by the fact that the three residues comprising the pocket are conserved in all chordate pol λ homologs (). Interestingly, two of the three residues are conserved in human pol μ, while only the charge of one of the three residues (Arg514) has been conserved in human pol β, as Lys 280.
Figure 2 Binding pocket for the scrunched nucleotide. a. Binding pocket for the scrunched nucleotide. The extrahelical nucleotide (yellow surface) is shown, together with the following and preceding template residues (red), the 5' residue in the downstream primer (more ...)
A triple mutant polymerase has decreased processivity
To test the functional significance of this template-binding pocket, we replaced the three conserved residues that contact the extrahelical nucleotide with alanine. Steady state kinetic analysis of single nucleotide gap filling demonstrated that, despite non-conservative replacement of three conserved residues, the catalytic efficiency of the purified triple mutant pol λ is only 5-fold lower than that of wild type pol λ(). Moreover, the triple mutant enzyme behaves like wild-type pol λ when copying a non-gapped primer-template, in that both enzymes are largely distributive when no downstream primer with a 5′ phosphate is present (, lanes 2 and 3). However, the results are different when filling a 5-nucleotide gap (, lanes 5 and 6). The total amount of product generated is greater than for simple primer extension (e.g., compare product band intensities in lane 3 versus 6), suggesting that the triple mutant still benefits from binding the downstream 5'-phosphate. As expected, wild type pol λ fills the gap (lane 5) more processively than for simple primer extension (lane 2). In contrast, gap filling DNA synthesis by the triple mutant is less processive (lane 6) than for wild type pol λ (lane 5), demonstrating that it dissociates from the DNA more readily and/or translocates less efficiently than wild type pol λ. When termination probabilities were calculated (see Methods) for each position as the gap was being filled, wild type and mutant pol λ behaved similarly for the first and second incorporations (, black versus gray bars at +1 and +2). In these instances, the numbers of uncopied template bases in the gap are four and three, respectively. However, at the +3 and +4 positions, the triple mutant enzyme terminated processive synthesis more often than did wild type pol λ. At the +4 position, which results from incorporation into a substrate where one uncopied base would need to be bound in the pocket, the difference in termination probability between the wild type and triple mutant is 5-fold. Thus, the effect of the pocket on processivity increases as the gap becomes shorter with the maximum effect seen on a two nucleotide gap with one nucleotide stabilized in the pocket.
Figure 3 a. Steady state kinetic analysis of nucleotide incorporation. Reactions were performed as described in Materials and Methods. b. Gap-filling activity assay. The wild type and the triple alanine mutant polymerases have comparable activity when polymerizing (more ...)
The binding pocket influences the fidelity of synthesis
The processivity data implies that the binding of an uncopied template base in the pocket contributes to the stability of the scrunched intermediate. Lower stability of a scrunched intermediate in the triple mutant predicts that it might affect the fidelity of DNA synthesis by pol λ, based on the following logic. Wild type pol λ is particularly prone to generate single base deletion intermediates during DNA synthesis23
. This property is thought to reflect its normal biological function, i.e., the ability to participate in NHEJ by filling short gaps formed when two broken ends are aligned with as little as a single correct terminal base pair7,23,24
. The mechanism for producing single base deletions is thought to involve dNTP-induced misalignment of the template-strand relative to the primer strand during catalytic cycling25
. This generates an intermediate with an extrahelical template nucleotide in the duplex DNA upstream of the polymerase active site24,26
. A wealth of evidence indicates that the ability of a polymerase to extend a misaligned intermediate to ultimately yield a single base deletion depends on the stability of the misaligned intermediate. Thus, as dNTP binding induces scrunching when filling gaps longer than a single nucleotide, the destabilization imparted by the triple mutant may disfavor extension of a misaligned intermediate more so than extension of an aligned intermediate, thereby reducing the rate at which pol λ generates single base deletions. To test this prediction, we compared the ability of wild type and triple mutant pol λ to generate single base deletions during synthesis to fill a 6-nucleotide gap containing a LacZ
template TTTT run23
(). As expected based on earlier studies23
, wild type pol λ is inaccurate (lacZ
mutant frequency 620 × 10−4
). In comparison, the triple mutant is 4-fold more accurate (lacZ
mutant frequency 160 × 10−4
). This is consistent with the idea that the triple mutant destabilizes scrunched intermediates in a manner that disfavors extension of misaligned template-primers.
Structural effects of the triple substitution
To further examine the role of the binding pocket for the second nucleotide in the gap, we obtained a 3Å crystal structure of the triple mutant using the same substrate used with wild-type pol λ (). Four molecules of the triple mutant are present in the asymmetric unit. Two of these are in a conformation similar to that observed in the wild-type pol λ structure. Scrunching is observed, i.e., the 3'-OH and 5'-PO4 groups maintain a similar distance to what is observed in a single-nucleotide gap. However, while the backbone of the template strand adopts the same conformation as in the wild type structure, the absence of the binding pocket results in an increase in the conformational flexibility of the base of the second nucleotide in the gap. The other two molecules in the asymmetric unit are in a conformation reminiscent of that observed in the original two-nucleotide structure (1RZT). The incoming dNTP is present, but the base is not bound in its usual conformation (see ), i.e., the protein is in the inactive conformation. This indicates that correct geometry between the incoming nucleotide and the templating base is required to transition to an active, scrunched structure, and suggests that the scrunched intermediate is less stable in the triple mutant than in wild type pol λ, an interpretation consistent with the biochemical data.
Figure 4 a. Scrunching in the triple alanine mutant. Aberrant nucleotide binding in the triple mutant structure. In two of the molecules in the asymmetric unit the nucleotide is only partly bound. As a consequence the polymerase has not adopted a pre-catalytic (more ...)
“Scrunching” and NHEJ
The ability of the polymerase to “scrunch” the template strand and conduct processive gap-filling could be important for NHEJ reactions when the ends to be joined contain gaps greater than a single nucleotide. To test this possibility, we performed NHEJ reactions with Ku heterodimer, XRCC4, DNA ligase IV and either wild type pol λ or the triple mutant pol λ. We used two different linear 280 base pair substrates with overhanging sequences such that end alignment results in either a 1-nucleotide or a 2-nucleotide gap (Substrates 1 and 2, respectively, ). These gaps must be filled by pol λ before concatemer ligation products are generated (e.g., , compare lanes 2 and 3). Using the substrate that requires filling a one-nucleotide gap, and where scrunching would not be necessary (Substrate 1), the triple mutant was slightly more efficient than wild type pol λ at promoting end-joining (, compare lanes 3 and 5). Thus the triple mutant enzyme is fully capable of performing one-nucleotide gap filling during NHEJ. In contrast, using a substrate requiring filling a two-nucleotide gap (Substrate 2), where the scrunching observed in the crystal structure would be relevant, the efficiency of end joining using the triple mutant pol λ was 8-fold lower than for wild type pol λ (, compare lanes 4 and 6). This indicates that the pocket that binds the uncopied template base is important for efficient NHEJ when end joining requires the filling of a 2-nucleotide gap.
Relevance of scrunching to longer gaps
The difference in termination by wild type and triple mutant pol λ seen at the +3 position as a 5-nucleotide gap is filled (), and the fact that the triple mutant is more accurate than wild type pol λ during synthesis to fill a 6-nucleotide gap () both suggest that the binding pocket for the second template nucleotide in the gap may also be relevant to filling gaps of at least three nucleotides. For this reason, efforts were made to obtain crystal structures involving 3-, 4- and 5-nucleotide gaps. These attempts all failed. This prompted three modeling studies using the scrunched dA structure () as an initial template. In one case, one dAMP was added 5' to the scrunched nucleotide. In another case, the dAMP was added 3' to the scrunched nucleotide. In the final case, two adenines were added 5' of the scrunched nucleotide. We then performed simulations (see Methods) on each of these models to investigate the position of the nucleotides in the gap throughout the simulation. In all cases, the simulations indicated that the nucleotide immediately 5' to the templating nucleotide is preferentially bound in the scrunching pocket, and that additional nucleotides 5' to this one could indeed be accommodated by the polymerase (). These models are consistent with the idea that pol
can accommodate additional uncopied template nucleotides while maintaining a conformation similar to that observed in the crystal structures. The models further suggest that, when given multiple choices, the scrunching pocket is more likely to accommodate the nucleotide immediately adjacent to the templating nucleotide.
Figure 5 Final conformation of the template strand after molecular dynamics simulations with a 4-nucleotide gap substrate. The single-stranded nucleotides in the gap are shown with their van der Waals surfaces colored. The distance between the C3′ atom (more ...)