Genes VII
13.5 Some DNA polymerases have a common structure |
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. Crystal structures show that there is a common type of arrangement in DNA polymerases in the E. coli pol I family. The T7 replication system (which belongs to this family) consists of a catalytic subunit coded by the phage, the bacterial protein thioredoxin (which increases processivity of the catalytic subunit), and two alternative products of phage gene 4, which provide primase and unwinding (helicase) activities.
| Figure 13.7 Crystal structure of phage T7 DNA polymerase has a right hand structure. DNA lies across the palm and is held by the fingers and thumb. Photograph kindly provided by Charles Richardson and Tom Ellenberger. |
| Figure 13.8 The catalytic domain of a DNA polymerase has a DNA-binding cleft created by three subdomains. The active site is in the palm. Proofreading is provided by a separate active site in an exonuclease domain. |
Figure 13.7 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 polymerase domain of the catalytic subunit has a structure that resembles a right hand. The existence of a large cleft composed of three domains is common to many DNA polymerases. The DNA lies across the palm in a groove that is created by the thumb and fingers. 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). Figure 13.8 shows the structure in diagrammatic form. The exonuclease domain responsible for proofreading is located below the palm, with a separate active site (this requires the 3’ end of the DNA chain to switch from the catalytic site to the exonuclease site when proofreading occurs).
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 13.7) rotated away from the palm. This suggests that an inward rotation of the O helix by ~41 X occurs to grasp the incoming nucleotide and create the active catalytic site, which is located in the palm subdomain at the bottom of the cleft. Amino acids in the active site contact the incoming base in such a way that the enzyme structure will be affected by a mismatched base; this may provide the signal that slows DNA synthesis and creates an opportunity for removal of the base by proofreading.
This section updated 1-27-2000