Skip Navigation

Your Environment. Your Health.

DNA Repair Enzymes

Structure Function Group

DNA Polymerases Lambda (λ) and Mu (μ) are members of the X Family, and are primarily involved in DNA repair. Both polymerases function in repair of DNA double strand breaks (DSBs), a particularly toxic type of lesion that can result in cell death if not corrected. In nonreplicating cells, these breaks are commonly repaired by a process known as nonhomologous end-joining (NHEJ). DSBs occur in a programmed manner during V(D)J recombination, a process that leads to maturation of immunoglobulin genes. However, DSBs also result from exposure to gamma radiation, reactive oxygen species, and exogenous environmental agents.

Pols λ and μ both function in NHEJ, though each polymerase is recruited to a specific type of broken DNA end. Pol λ has a strict requirement for a paired primer terminus. In contrast, Pol μ is the only known eukaryotic polymerase that can act on broken DNA ends with no sequence complementarity at the site of the break, i.e., synapses involving an unpaired primer terminus.

Our lab employs X-ray crystallography to understand the structural determinants of DNA binding, specificity and catalysis by these polymerases. To this end, we have solved crystal structures in complex with a variety of DNA substrates. Nonhydrolyzable analogs of naturally occurring nucleotides are used to capture catalytically relevant complexes on the cusp of the enzymatic reaction. Once the crystals have formed, we can remove such analogs, and replace them with the catalytically competent nucleotide, which allows the reaction to run to completion in the crystal.  

Our structures have revealed that there is a gradient of protein and DNA substrate movements that are essential for efficient catalysis by the Family X polymerases. Pol β, with the strictest substrate requirements, undergoes large-scale motions of protein subdomains, DNA substrate, and active site side chains during catalysis. Pol λ, with a slightly more flexible DNA substrate specificity, forgoes global protein subdomain motions, but uses more subtle adjustments of DNA template and active site chains to achieve optimal positioning. By contrast, Pol μ functions as a rigid scaffold, and having few requirements for DNA template strand structure. Rigidity of the protein appears to allow for flexibility of template strand conformation or distortion.  Additionally, our structures reveal that, while most gap-filling polymerases use the upstream side of the gap (closest to the 3ʹ-OH on the primer terminus) to direct synthesis, Pol μ instead ‘skips ahead’, using the downstream end of the gap (closest to the 5ʹ-phosphate) as a guide. This ‘skip ahead’ mechanism appears to be a unique to Pol μ and is consistent with the reported behavior of this enzyme in vivo.  

Due to the complexity of creating a DNA synapsis of noncomplementary ends — using only the polymerase to bridge the gap — our studies thus far have focused on the more simplistic system of single strand DNA breaks.  However, we strive to structurally characterize the behavior of the Family X polymerases in DSB repair, and to understand the intermolecular interactions involved in the context of the larger NHEJ complex machinery.  

Structural characterization of human DNA Polymerase
Figure 1:  Structural characterization of human DNA Polymerase μ with either a 1- (left) or 2-nucleotide (right) gapped single-strand break DNA substrate. These structures show that Pol μ functions as a rigid scaffold (left), since there are no large-scale movements of either DNA substrate, protein subdomains, or active site side chains, from DNA binding (binary complex with protein in blue, DNA in light blue), nucleotide binding (pre-catalytic ternary complex with protein in orange, DNA in khaki), through catalysis (post-catalytic nicked complex with protein in red, DNA in pink).  When bound to a gap longer than a single nucleotide (right), Pol μ uses the last available nucleotide in the gap (+1 position), rather than the first (0 position), as a guide. The enzyme accommodates the unpaired template nucleotide within the active site, with minimal distortion.