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  • In the case of high fidelity polymerases we propose


    In the case of high-fidelity polymerases, we propose that water is used to achieve negative selection against nucleotide binding. With specialized DNA polymerases, we propose that nucleobase desolvation plays a different yet important role in allowing these enzymes to replicate damaged DNA. In this model, specialized DNA polymerases use enthalpic–entropic compensation as a way to generate high catalytic efficiency during TLS. This is possible as the expanded active site of a specialized NS 1619 synthesis is large enough to bind a fully solvated nucleotide. This essentially bypasses the initial requirement for nucleobase desolvation. Consistent with this mechanism are previous data obtained using the specialized DNA polymerase, pol η, in which the Km values measured for modified and artificial nucleotides were independent of their hydrophilic or hydrophobic nature [37]. For instance, the Km for dATP is 47μM, while the Km values for hydrophobic analogs such as N6-Me-dATP, O6-Me-dGTP, 5-EyITP, and 5-NITP remain invariant at ~50μM [37]. While this mode of binding may seem counterproductive, the ability of specialized DNA polymerases to bind a fully solvated nucleotide could play two important roles in its primary function to replicate damaged DNA. First, water molecules surrounding the incoming nucleobase could move within the active site of the polymerase. This mobility could generate greater flexibility that could subsequently provide these DNA polymerases opportunities to optimize productive interactions between the incoming nucleotide and a DNA lesion. In this model, the greater entropy provided by increased water mobility may allow these polymerases to accommodate a variety of structurally distinct DNA lesions ranging from non-instructional including abasic sites to crosslinked lesions such as thymine dimers and cisplatinated DNA. In addition, the larger solvation sphere could also provide enthlapic stabilization by increasing hydrogen-bonding interactions that are required for interactions between the incoming nucleotide and a DNA lesion. The synergy NS 1619 synthesis between these features may account for improved efficiency of specialized polymerases such as pol eta to perform TLS. In addition, this may also explain why certain specialized DNA polymerases display reduced fidelity when replicating undamaged DNA. In this case, the ability of the polymerase to move water molecules within its active site could allow the polymerase to form mispairs more easily. While the kinetic studies described here provide initial evidence redefining the role of nucleobase desolvation during replication, we acknowledge that more experimentation is needed to fully quantify this biophysical feature during normal and translesion DNA synthesis. Toward this goal, we are currently examining the effects of molecular crowding agents and solvent isotope effects to further explore the role of nucleobase desolvation by both high-fidelity and specialized DNA polymerases.
    Material and Methods
    Acknowledgments The authors wish to thank Dr. Jung-Suk Choi for helpful discussions and critical reading of this manuscript. Research was funded by the Department of Defense (W81XWH-13-1-0238) to A.J.B. and a Dissertation Research Award (0010-1710-10 DDDSARI) provided by Cleveland State University to A.D.
    Introduction The continued proliferation of any cell requires that the genetic material be passed onto the next generation of cells in an undamaged state. In human cells, the genetic information is stored in 23 pairs of chromosomes (22 pairs of autosomes and 1 pair of sex chromosomes). The replication and segregation of these 46 chromosomes are highly regulated during the cell division cycle. This cycle comprises interphase, when the cells grow in size and the chromosomes are replicated, and M-phase when the chromosomes and cytoplasm are segregated equally to the two daughter cells. Mitosis is subdivided into five subphases: prophase, prometaphase, metaphase, anaphase, and telophase. Until recently, the conventional view of the cell cycle was that DNA is fully replicated during interphase otherwise entry into mitosis would be prohibited. However, this view has been challenged recently by the finding that some regions of the human genome seem only to be fully replicated in the early stages of mitosis. These “tardy” regions are not randomly distributed in the genome, but instead concentrate at loci that are known as common fragile sites (CFSs) (Minocherhomji et al., 2015). They are referred to as being “fragile” because they lie within chromosomal loci that have a propensity to appear as a gap or break on otherwise fully condensed metaphase chromosomes. This phenomenon is usually termed CFS expression.