Klenow fragment structure

A detailed analysis of solid-state structures of several unrelated systems that catalyze the hydrolysis of diphosphate esters (alkaline phosphatase and the Klenow fragment , among others) revealed that their active sites invariably contain conserved carboxylate... 

DNA polymerase I, then, is not the primary enzyme of replication instead it performs a host of clean-up functions during replication, recombination, and repair. The polymerase s special functions are enhanced by its 5 —>3 exonuclease activity. This activity, distinct from the 3 —>5 proofreading exonuclease (Fig. 25-7), is located in a structural domain that can be separated from the enzyme by mild protease treatment. When the 5 —>3 exonuclease domain is removed, the remaining fragment (Afr 68,000), the large fragment or Klenow fragment (Fig. 25-8), retains the polymerization and... 

Some bacteriophage encode their own DNA polymerases. However, they usually rely on the host cell to provide accessory proteins. The sequence of the DNA polymerase from phage T7 is closely homologous to that of the Klenow fragment and the 3D structures are similar. The 80-kDa T7 polymerase requires the 12-kDa thioredoxin from the host cell as an additional subunit. It has been genetically engineered to improve its usefulness in DNA sequencing 278... 

Beese, L. S., V. Derbyshire, and T. A. Steitz, Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260 352-355, 1993. 

Girard PM, D Flam C, Cadet J, Boiteux S (1998) Opposite base-dependent excision of 7,8-dihydro-8-oxo-adenine by the Oggl protein of Saccharomyces cerevisiae. Carcinogenesis 19 1299-1305 Greenberg MM, Matray TJ (1997) Inhibition of Klenow fragment (exo ) catalyzed DNA polymerization by (5.R)-5,6-dihydro-5-hydroxy thymidine and structural analogue 5,6-dihydro-5-methyl-thymidine. Biochemistry 36 14071-14079... 

Beese, L. S., Friedman, J. M., and Steitz, T. A. (1993b). Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Biochemistry 32, 14095-14101. 

Li, Y., Kong, Y., Korolev, S., and Waksman, G. (1998a). Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleo-side triphosphates. Protein Sci. 7, 1116-1123. 

Because of the hairpin formation, these dyes are in such a close proximity that their fluorescence is quenched (molecular beacon Box 18) unless the structure is unfolded in the course of second-strand synthesis (Figure 4.3.4b). Thus, detection of a fluorescence signal from one of both dyes is a direct measure of the progress of the reaction. These researchers also showed that primer extension reactions can be monitored directly in cleared lysates of cells overexpressing the Klenow fragment of E. coli DNA polymerase I. Thus, the molecular beacon assay might supersede extensive purification. 

Figure 12. Tertiary structure of the Klenow fragment of E. c,oZi DNA polymerase I, based on X-ray crystallographic data. The a-helices are represented by tubes (lettered) and 3-sheets by arrows (numbered). (Reproduced with permission from Ref. 23. Copyright 1987 Alan R. Liss, Inc.)...

P. N. S. Yadav, J. S. Yadav, and M. J. Modak, Biochemistry, 31, 2879 (1992). A Molecular Model of the Complete Three-Dimensional Structure of the Klenow Fragment of Escherichia coli DNA Polymerase I Binding of the dNTP Substrate and Template-Primer. 

The three-dimensional structures of a number of DNA polymerase enzymes are known. The first such structure to be determined was that of the so-called Klenow fragment of DNA polymerase I from E. coli (Figure 27.11). This fragment comprises two main parts of the full enzyme, including the polymerase unit. This unit approximates the shape of a right hand with domains that are referred to as the fingers, the thumb, and the palm. In addition to the polymerase, the Klenow... 

Figure 27.11. DNA Polymerase Structure. The first DNA polymerase structure determined was that of a fragment of E. coli DNA polymerase I called the Klenow fragment. Like other DNA polymerases, the polymerase unit resembles a right hand with fingers (blue), palm (yellow), and thumb (red). The Klenow fragment also includes an exonuclease domain.

It has been shown that the G3-induced deletions are efficient in both E. coli Pol II exo" and exonuclease-free Klenow fragments [87]. These results imply a common AAF-induced SMI structure in the Narl sequence. The suggestion is that such a... 

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