The quick and accurate replication of genetic information (DNA) is critical to all life. DNA polymerase, a key enzyme for DNA replication in organisms, catalyzes the reaction that extends the primer strand of DNA during replication. Before catalysis, DNA polymerase has the daunting task of matching a template nucleic acid base with its corresponding Watson-Crick base (dNTP) that must be added to the primer strand. The enzyme must have high specificity in order to distinguish one correct base for incorporation from three incorrect bases. Crystal structures of DNA polymerase have shown an important a-helix on the enzyme, called the O-helix, plays a crucial role in binding the incoming dNTP. The O-helix forms an �open� conformation in the absence of a dNTP in the active site of DNA polymerase, �closed� when a proper Watson-Crick base pair is matched, and is relatively stable in an intermediate �ajar� state in the event a mismatched dNTP enters the active site. In this study, we have utilized recent computational advances to simulate the opening of the fingers domain starting from the closed and ajar conformations of Bacillus stearothermophilus DNA polymerase Klenow fragment using dynamics on the microsecond time scale, and also simulated the open state for comparison. We have fully characterized the opening process that occurs after catalysis, and determined the key events and movements that are critical to O-helix opening. The transition from ajar to open is quick,while the full transition from closed to open was significantly longer. We observed a key intermediate in the pathway from closed to open involving a salt bridge between an arginine side chain on the O-helix and an aspartate in the thumb domain. These simulations aid in the elucidation of the O-helix opening mechanism for DNA polymerase on an atomistic level not currently available with experimental measures.

Investigation into the opening mechanism for DNA polymerase using molecular dynamics simulations

248th National Meeting (2014)

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