The Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA

 

The Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA

 

Background:
KLENOW FRAGMENT of DNA POL I
by, Sundeep Kang
DNA Pol I consists of a single polypeptide, 928 redisues in lengh. Pol I was the first DNA polymerase discovered, and it was thought to be responsible for the DNA replication. Pol I links two deoxynucleoside triphosphates; the second dNTP must be linked to the free 3-OH of the previous nucleoside. The release and subsequent hydrolysis of PPi drives the process, and the specificity of the which nucleoside will be added to the growing polynucleoside chain results from the fact that the incoming dNTP must form Watson-Crick base pairs with the template strand of DNA from which the copy is made. The entire Pol I enzyme consists of polymerase activitity, in addition to 5`-3` and 3`-5` exonuclease activity. All three occupy different active sites on the enzyme, however when proteases are added the enzyme breaks down into two fragments. The larger fragment known as the Klenow Fragment is about 604 residues contains the polymerase active site as well as the 3`-5` exonuclease active site. This Klenows was crystallized in "editing mode", where the protein exhibits exonuclease activity. When the protein is "polymerizing mode" the polymerae site is active. (Klenow backbone)
The resulting 324 residue smaller fragment contains the 5`-3` exonuclease site. The Klenow frgament serves various purposes in molecular biology, including use in DNA sequencing and radioactive labeling experiments. The Klenow Fragment consists of two domains. The small domain (residues 324-517) contains the 3`-5` exonuclease site (Small domain)
The larger domain (528-928) contains the polymerase active site. The polymerase site can be seen as being located at the bottom the verticle cleft on the top the molecule. This cleft is lined with positively charged molecules, is about 22 A wide and 30 A deep, and forms what is known as the "finger and thumb" of the molecule. (Large domain)
Specificly,Helices H and I ( right of top cleft) forms what is refered to as the "thumb" of the molecule. Helixes L-P (left of the large top cleft) are known as the "Fingers" (Secondary structure)
This crystal structure shows the Klenow in the "editing mode". However when the Klenow is in the polymerizing mode, there are highly conserved molecules which participtate in polymerization. Previous experiments which dealt with dNTP binding to the polymerase site revealed that incoming dNTP's interact with Arg 754, Arg 682,Lys 758, His 734 . These residues are highly conserved in the family of DNA polymerases, and interact with the phosphate backbone of incoming dNTPs. (positive residues which line large cleft)
These conserved residues are all positivaly charged and line the large polymerase cleft. Closer examination reveals that there are a number of other conserved residues which participate in the incorporation of incoming dNTP with a negatively charged phosphate backbone. Previous experiments and x-ray data have shown that Tyr 766, Asp 882, Asp 705, Glu 883, and His 881 are all involved in binding and directing the incoming dNTP (dNTP binders in polymerase site)
This Klenow Fragment, in editing mode was cocyrstalized with and an 11 bp duplex DNA. Notice that the DNA is not near the polymerase site in editing complex, rather it located in the exonuclease site near the palm of the molecule. The exonuclease site and the polymerase site are about 35 A apart. (DNA backbone)
At the exonuclease site there is a ss 3nt overhang of DNA, which is the primer strand. (3nt primer on DNA)
This crystal structure reveals that the primer strand enters the polymerase site throught the exonuclease, and not throught the top of the large cleft. However when the Klenow shifts between exonuclease mode and polymerase mode, it is the 3nt overhang on the primer that moves from the exonuclease site to the polymerase site . However, because the two clefts are approxmiately perpedicular to each other it is clear that the duplex DNA must bend in order to go to polyermzing mode. Three very important residues in the polymerase cleft which faciliate this process are Asp 882, Glu 883, and Asp 705. These redisidues bind to divalent cations and enable the duplex DNA to bend toward the polymerase active site where incoming dNTPs can replicate the template strand. (cat. important aa polymerase site)
The primer and DNA enter through the exonuclease site, not the large cleft, as previously thought. Previous mutation studies of exonuclease site have shown that Leu 361, Phe 473, His 660, and Glu 357 are significantly involved with primer interaction and proofreading activity through the help of divalent cations and hydrophobic interactions. (aa in exonuclease site)
Since the DNA enters through the exonuclease site to reach the polymerase site, there are residues that are located on the thumb of the molecule which are directly involved in binding the DNA. These amino acids are highly conserved: Lys 635,Asn 678, and Asn 675 only interact with the backbone of the DNA primer strand. (DNA binding residues on "thumb")
Based on previous experiments the "thumb regions goes through a conformational change and makes direct contact with duplex DNA by moving closer to the exonuclease site. Thus DNA causes a conformational change in the Klenow; when duplex DNA is present it resides in the new second cleft, which is perpindicular to the orginal top cleft. In addition to positively charged residues which contact the primer strand backbone, other conserved residues such as Arg 835, Asp 827, Ser 582, and Asn 579 also make interactions with the template strand backbone to stabalize the duplex inside the Klenow. Once duplex DNA is bound ( as shown in this structure) the protein makes contact only with the DNA backbone. (DNA binding AA)
In summation the Klenow fragment exists in two different conformational forms when polymerase activity if required and when exonuclease activity is required. However it is clear from highly conserved amino acids that the DNA/primer strand cause a conformatational change which produces a new cleft near the exonuclease site.The DNA enters through this cleft and then the moving primer strand moves(along with a conformational change in the protein) to the polymerise site, which also contains positive residues which interact with the duplex backbone. The presences of this intricate system increases Pol I's fidelity during replication. References: L.Beese et al., "The Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA",Science 260(1993) L.Beese et al.,"Crystal Structure of the Klenow Fragment of DNA Pol I....", Biochemistry 32 (1993) W.Lam et al., "Effects of Mutations on...", Biochemistry 37 (1998) V.Derbyshire et al., " Genetic and Crystallographic Studies of 3'and 5' Exonucleolytic Site of Pol I", Science 240 (1993)