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Key Terms
  • Many DNA polymerases have a large cleft composed of three domains that resemble a hand.
  • DNA lies across the "palm" in a groove created by the "fingers" and "thumb."

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Figure 14.7  
The common organization of DNA polymerases has a palm that contains the catalytic site, fingers that position the template, a thumb that binds DNA and is important in processivity, an exonuclease domain with its own active site, and an N-terminal domain.

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Figure 14.7 shows that all DNA polymerases share some common structural features (3091; 3092). The enzyme structure can be divided into several independent domains, which are described by analogy with a human right hand. DNA binds in a large cleft composed of three domains. The "palm" domain has important conserved sequence motifs that provide the catalytic active site. The "fingers" are involved in positioning the template correctly at the active site. The "thumb" binds the DNA as it exits the enzyme, and is important in processivity. The most important conserved regions of each of these three domains converge to form a continuous surface at the catalytic site. The exonuclease activity resides in an independent domain with its own catalytic site. The N-terminal domain extends into the nuclease domain. DNA polymerases fall into five families based on sequence homologies; the palm is well conserved among them, but the thumb and fingers provide analogous secondary structure elements from different sequences.

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Figure 14.8  
The crystal structure of phage T7 DNA polymerase shows that the template strand takes a sharp turn that exposes it to the incoming nucleotide. Photograph kindly provided by Charles Richardson and Tom Ellenberger (see 3748).

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The catalytic reaction in a DNA polymerase occurs at an active site in which a nucleotide triphosphate pairs with an (unpaired) single strand of DNA. The DNA lies across the palm in a groove that is created by the thumb and fingers. Figure 14.8 shows the crystal structure of the T7 enzyme complexed with DNA (in the form of a primer annealed to a template strand) and an incoming nucleotide that is about to be added to the primer. The DNA is in the classic B-form duplex up to the last 2 base pairs at the 3′ end of the primer, which are in the more open A-form. A sharp turn in the DNA exposes the template base to the incoming nucleotide. The 3′ end of the primer (to which bases are added) is anchored by the fingers and palm. The DNA is held in position by contacts that are made principally with the phosphodiester backbone (thus enabling the polymerase to function with DNA of any sequence).

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In structures of DNA polymerases of this family complexed only with DNA (that is, lacking the incoming nucleotide), the orientation of the fingers and thumb relative to the palm is more open, with the O helix (O, O1, O2; see Figure 14.8 rotated away from the palm. This suggests that an inward rotation of the O helix occurs to grasp the incoming nucleotide and create the active catalytic site. When a nucleotide binds, the fingers domain rotates 60° toward the palm, with the tops of the fingers moving by 30 Å. The thumb domain also rotates toward the palm by 8°. These changes are cyclical: they are reversed when the nucleotide is incorporated into the DNA chain, which then translocates through the enzyme to recreate an empty site.

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The exonuclease activity is responsible for removing mispaired bases. But the catalytic site of the exonuclease domain is distant from the active site of the catalytic domain. The enzyme alternates between polymerizing and editing modes, as determined by a competition between the two active sites for the 3′ primer end of the DNA (3093). Amino acids in the active site contact the incoming base in such a way that the enzyme structure is affected by a mismatched base. When a mismatched base pair occupies the catalytic site, the fingers cannot rotate toward the palm to bind the incoming nucleotide. This leaves the 3′ end free to bind to the active site in the exonuclease domain, which is accomplished by a rotation of the DNA in the enzyme structure (3094).

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reviews
  • 3091 Joyce, C. M. and Steitz, T. A. (1994).  Function and structure relationships in DNA polymerases.  Annu. Rev. Biochem. 63, 777-822.  PubMed   Journal
  • 3092 Hubscher, U., Maga, G., and Spadari, S. (2002).  Eukaryotic DNA polymerases.  Annu. Rev. Biochem. 71, 133-163.  PubMed   Journal
  • 3093 Johnson, K. A. (1993).  Conformational coupling in DNA polymerase fidelity.  Annu. Rev. Biochem. 62, 685-713.  PubMed   Journal
reviews
  • 3094 Shamoo, Y. and Steitz, T. A. (1999).  Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex.  Cell 99, 155-166.  PubMed   Journal

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