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One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson–Crick base pair with correct deoxycytidine 5′-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5′-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson–Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg2+ appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3′-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion.

Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyltransfer (i.e., chemistry), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallographic studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidence for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.

Assimilate supply to the developing ear of maize (Zea mays L.) is an important determinant of grain yield. The objective of the current study was to determine the relative limitations of photosynthate and reduced N supply to the ear for determination of yield components, kernel number and kernel weight. Field‐grown maize plants on Dupo silt loam (Coarse‐silty over clayey, mixed, nonacid, mesic Aquic Udifluvents) were shaded during either vegetative growth, flowering, or grain fill. Control plants were not shaded. Photosynthesis was measured on plots from 9 d before flowering to grain maturity, and plants were sampled at intervals during this period for measurement of dry weight and reduced N content of plant parts of the aboveground vegetation (stover) and ear. When plants were shade during flowering, photosynthesis decreased during this period and kernel abortion increased relative to controls. However, N concentration was higher in aborting kernels than in nonaborting kernels through late flowering and early grain fill. The supply of reduced N to the ear during flowering was not a limiting factor for determination of kernel number. During grain fill, remobilization of N and dry matter from the stover of controls accounted for 46.5 and 4.7% of ear N and dry weight at maturity, respectively. Availability of newly reduced N was apparently more limiting than availability of current photosynthate for kernel dry weight accumulation. It is proposed that supply of newly reduced N to the ear may be limited by the amount of photosynthate partitioned for nitrate uptake and reduction during grain fill.

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