DNA pol λ's extraordinary ability to stabilize misaligned DNA.

DNA polymerases have the venerable task of maintaining genome stability during DNA replication and repair. Errors, nonetheless, occur with error propensities that are polymerase specific. For example, DNA polymerase λ (pol λ) generates single-base deletions through template-strand slippage within short repetitive DNA regions much more readily than does the closely related polymerase ß (pol ß). Here we present in silico evidence to help interpret pol λ's greater tendency for deletion errors than pol ß by its more favorable protein/DNA electrostatic interactions immediately around the extrahelical nucleotide on the template strand. Our molecular dynamics and free energy analyses suggest that pol λ provides greater stabilization to misaligned DNA than aligned DNA. Our study of several pol λ mutants of Lys544 (Ala, Phe, Glu) probes the interactions between the extrahelical nucleotide and the adjacent Lys544 to show that the charge of the 544 residue controls stabilization of the DNA misalignment. In addition, we identify other thumb residues (Arg538, Lys521, Arg517, and Arg514) that play coordinating roles in stabilizing pol λ's interactions with misaligned DNA. Interestingly, their aggregate stabilization effect is more important than that of any one component residue, in contrast to aligned DNA systems, as we determined from mutations of these key residues and energetic analyses. No such comparable network of stabilizing misaligned DNA exists in pol ß. Evolutionary needs for DNA repair on substrates with minimal base-pairing, such as those encountered by pol λ in the non-homologous end-joining pathway, may have been solved by a greater tolerance to deletion errors. Other base-flipping proteins share similar binding properties and motions for extrahelical nucleotides.

Relationship between conformational changes in pol lambda's active site upon binding incorrect nucleotides and mismatch incorporation rates.

The correct replication and repair of DNA is critical for a cell's survival. Here, we investigate the fidelity of mammalian DNA polymerase lambda (pol lambda) utilizing dynamics simulation of the enzyme bound to incorrect incoming nucleotides including A:C, A:G, A(syn):G, A:A, A(syn):A, and T:G, all of which exhibit differing incorporation rates for pol lambda as compared to A:T bound to pol lambda. The wide range of DNA motion and protein residue side-chain motions observed in the mismatched systems demonstrates distinct differences when compared to the reference (correct base pair) system. Notably, Arg517's interactions with the DNA template strand bases in the active site are more limited, and Arg517 displays increased interactions with the incorrect dNTPs. This effect suggests that Arg517 helps provide a base-checking mechanism to discriminate correct from incorrect dNTPs. In addition, we find Tyr505 and Phe506 also play key roles in this base checking. A survey of the electrostatic potential landscape of the active sites and concomitant changes in electrostatic interaction energy between Arg517 and the dNTPs reveals that pol lambda binds incorrect dNTPs less tightly than the correct dNTP. These trends lead us to propose the following order for mismatch insertion by pol lambda: A:C > A:G > A(syn):G > T:G > A(syn):A > A:A. This sequence agrees with available kinetic data for incorrect nucleotide insertion opposite template adenine, with the exception of T:G, which may be more sensitive to the insertion context.

Substrate-induced DNA strand misalignment during catalytic cycling by DNA polymerase lambda.

The simple deletion of nucleotides is common in many organisms. It can be advantageous when it activates genes beneficial to microbial survival in adverse environments, and deleterious when it mutates genes relevant to survival, cancer or degenerative diseases. The classical idea is that simple deletions arise by strand slippage. A prime opportunity for slippage occurs during DNA synthesis, but it remains unclear how slippage is controlled during a polymerization cycle. Here, we report crystal structures and molecular dynamics simulations of mutant derivatives of DNA polymerase lambda bound to a primer-template during strand slippage. Relative to the primer strand, the template strand is in multiple conformations, indicating intermediates on the pathway to deletion mutagenesis. Consistent with these intermediates, the mutant polymerases generate single-base deletions at high rates. The results indicate that dNTP-induced template strand repositioning during conformational rearrangements in the catalytic cycle is crucial to controlling the rate of strand slippage.

Simulations of DNA pol lambda R517 mutants indicate 517's crucial role in ternary complex stability and suggest DNA slippage origin.

Unlike some other DNA polymerases, DNA polymerase lambda (pol lambda) utilizes DNA motion and active-site protein residue rearrangements rather than large-scale protein subdomain changes to transition between its active and inactive states. Pol lambda also has an unusual error tendency to generate single-base deletions (also known as frameshift mutations) resulting from DNA template-strand slippage. An understanding of these features requires an atomic-level link between the various structures and motions involved and observed in biochemical functions. Our simulations of pol lambda ternary complexes of various 517 mutants (Lys, Glu, His, Met, and Gln) reveal discrete orientations of the 517 residue with respect to the DNA and associated interactions (mainly electrostatic) that explain the wide range ( approximately 3-8 A) of mutant-dependent DNA motion observed (Figure 2 of manuscript): (wild-type < [R517K approximately R517H approximately R517Q] < [R517E approximately R517A approximately R517M]). This motion critically impacts stability of the ternary complex and hence drives/hampers the enzyme's catalytic cycle. In addition to pinpointing a trend for interpreting associated frameshift error rates based on template-strand stability, the close connection between DNA movement and active-site protein residue changes suggests that pol lambda's unique architecture facilitates frameshift errors because small variations in the active-site environment (e.g., orientation of 517) can have large effects on the dynamics of the ternary pol lambda complex.

Sequential side-chain residue motions transform the binary into the ternary state of DNA polymerase lambda.

The nature of conformational transitions in DNA polymerase lambda (pol lambda), a low-fidelity DNA repair enzyme in the X-family that fills short nucleotide gaps, is investigated. Specifically, to determine whether pol lambda has an induced-fit mechanism and open-to-closed transition before chemistry, we analyze a series of molecular dynamics simulations from both the binary and ternary states before chemistry, with and without the incoming nucleotide, with and without the catalytic Mg(2+) ion in the active site, and with alterations in active site residues Ile(492) and Arg(517). Though flips occurred for several side-chain residues (Ile(492), Tyr(505), Phe(506)) in the active site toward the binary (inactive) conformation and partial DNA motion toward the binary position occurred without the incoming nucleotide, large-scale subdomain motions were not observed in any trajectory from the ternary complex regardless of the presence of the catalytic ion. Simulations from the binary state with incoming nucleotide exhibit more thumb subdomain motion, particularly in the loop containing beta-strand 8 in the thumb, but closing occurred only in the Ile(492)Ala mutant trajectory started from the binary state with incoming nucleotide and both ions. Further connections between active site residues and the DNA position are also revealed through our Ile(492)Ala and Arg(517)Ala mutant studies. Our combined studies suggest that while pol lambda does not demonstrate large-scale subdomain movements as DNA polymerase beta (pol beta), significant DNA motion exists, and there are sequential subtle side chain and other motions-associated with Arg(514), Arg(517), Ile(492), Phe(506), Tyr(505), the DNA, and again Arg(514) and Arg(517)-all coupled to active site divalent ions and the DNA motion. Collectively, these motions transform pol lambda to the chemistry-competent state. Significantly, analogs of these residues in pol beta (Lys(280), Arg(283), Arg(258), Phe(272), and Tyr(271), respectively) have demonstrated roles in determining enzyme efficiency and fidelity. As proposed for pol beta, motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.