STRUCTURE AND MECHANISM OF DNA POLYMERASES By PAUL J. ROTHWELL AND GABRIEL WAKSMAN Institute of Structural Molecular Biology, University College London and Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom I. Introduction ........................................................... 402 II. Biological Diversity of DNA Polymerases ................................ 403 A. Family A ........................................................... 403 B. Family B ........................................................... 403 C. Family C ........................................................... 404 D. Family D ........................................................... 404 E. Family X ........................................................... 404 F. Family Y ........................................................... 405 G. RT Family ......................................................... 406 III. The Nucleotide Incorporation Pathway ................................. 406 A. General Theme .................................................... 406 B. Variation on a Theme .............................................. 408 IV. The E State: Basic Architecture of DNA Polymerases .................... 409 V. Primer/Template DNA Binding and Recognition ....................... 414 VI. Formation of the E:p/t:dNTP Complex ................................. 417 VII. Conformational Transition to a Catalytically Active Ternary Complex: The E’:p/t:dNTP Complex ................................... 419 A. Structures of E’:p/t:ddNTP Complexes ............................. 420 B. Molecular Basis for the Rate‐Limiting Step.......................... 423 C. Discrimination Between Correct Versus Incorrect Nucleotide ........ 426 VIII. Phosphoryl Transfer Reaction, Product Release, and Translocation of the Primer/Template DNA .......................................... 428 A. Phosphoryl Transfer Reaction and Product Release ................. 428 B. Translocation of Primer/Template DNA ............................ 429 C. Force Generation: DNA Polymerases as Molecular Motors ........... 429 IX. Conclusion............................................................. 430 References ............................................................. 431 Abstract DNA polymerases are molecular motors directing the synthesis of DNA from nucleotides. All polymerases have a common architectural frame- work consisting of three canonical subdomains termed the fingers, palm, and thumb subdomains. Kinetically, they cycle through various states corresponding to conformational transitions, which may or may not gener- ate force. In this review, we present and discuss the kinetic, structural, and single‐molecule works that have contributed to our understanding of DNA polymerase function. ADVANCES IN 401 Copyright 2005, Elsevier Inc. PROTEIN CHEMISTRY, Vol. 71 All rights reserved. DOI: 10.1016/S0065-3233(04)71011-6 0065-3233/05 $35.00