I. Introduction
II. Pol B Domain Structure:

• 8-kD Domain
  
• The Thumb
  
• The Palm
  

III. Catalytic Site and Reaction
IV. Additional Structure-Function Relationships:

• DNA Binding
  
• Processivity
  
• Fidelity

V. References

Note:  This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.

DNA polymerase Beta (pol B) is a eukaryotic DNA polymerase studied most extensively in vertebrate systems. Like other DNA polymerases, pol B is responsible for adding new nucleotides to a growing chain by catalyzing a nucleotidyl transfer reaction. Pol B is not the main DNA polymerase in eukaryotes; it is primarily involved in DNA repair. It has the unique ability to repair single-stranded DNA gaps smaller than 6 nucleotides.  Pol B is a single polypeptide chain of 335 amino acids.  It has no exonuclease (proofreading) activity, unlike some other DNA polymerases. Pol B's relatively simple structure lends itself to nucleotidyl transfer studies.

This exhibit explores structural aspects of Pol B catalysis, DNA binding, processivity, and fidelity. Before delving into these subjects, however, let's first study the basic architecture of the Pol B enzyme.

Pol B consists of two domains, an 8 kD domain and a 31 kD domain .   The 31 kD domain consists of several subdomains: the fingers, the thumb, and the palm .   All of these domains are necessary for catalysis.

8-kD Domain
The NH2-terminal 8 kD domain has been shown to bind single-stranded (ss) DNA (K_d = 2 x 10⁵ M^-1), and is thought to participate in template binding. This domain is composed of two pairs of antiparallel helices . The domain is highly basic (with a net charge of +10).  There is no structural equivalent to the 8 kD domain in other DNA polymerases, suggesting that it is involved with the special short gap filling activities associated with Pol B. One idea is that the 31 kD domain binds to dsDNA and the 8 kD domain binds to the ss template overhang.

The Thumb
Pol B changes conformation to clasp DNA, with considerable movement of the thumb subdomain. The thumb contains the COOH-terminus, three a helices and a two-stranded b-sheet .  The thumb has the ability to change its position by as much as 12 Å, closing or opening the active site. Most of this movement occurs because of flexible torsion angles in a hinge loop connecting the palm and thumb subdomains . Movement of the thumb is constrained by extensive contacts between beta strands 1,2 and 5 of the palm, and helix M of the thumb .   This interface between the thumb and palm is lined with hydrophobic groups on either side of the hinge so that regardless of whether the thumb is open or closed, there are an equal number of favorable hydrophobic interactions. There are four hydrophobic residues on the inside of the hinge (leucine, isoleucine, phenylalanine, and tyrosine), and four on the outside (isoleucine, threonine, tyrosine, and valine).

In addition to these hydrophobic interactions, there are two salt bridges, one between an arginine and glutamic acid on the inside of the hinge and one between another arginine and glutamic acid on the outside . Also, there are two hydrogen bonds, present only when the thumb is in the closed position, between backbone carbonyl oxygens and amide nitrogens that are located on the inside part of the hinge .

The Palm
The palm is the most structurally conserved subdomain . It contains two a-helices packed against the face of a b-sheet . This common motif is termed a "two layered alpha-beta sandwich". a-b sandwich motifs have high affinity for phosphate groups, and this likely plays a key role in Pol B's attraction to the DNA backbone. 

The DNA binding channel in Pol B is lined with positively charged lysine and arginine side chains that function to stabilize the negatively charged backbone of the DNA . The Pol B catalytic site contains three key aspartate residues . Asp²⁵⁶ is involved primarily with stabilizing the transition state complex and asp¹⁹⁰ and asp¹⁹² both aid in positioning an incoming nucleotide. These residues are complexed with two magnesium ions that are essential in binding the incoming nucleotide . In the crystal structure shown, the incoming nucleotide is a dideoxynucleotide (ddCTP), used to "freeze" the polymerase so that co-crystals of template, primer and polymerase could be obtained. The three catalytic asp residues have been modeled to go through several conformational changes during catalysis.

The magnesium ions have different functions in catalysis. One Mg²⁺ ion binds specifically to the beta and gamma phosphates of the ddNTP as a bidentate . The other Mg²⁺ acts to stabilize the negative a-phosphate of the ddNTP . The metal ions are an essential component of the overall reaction for positioning, stabilization, and activation.

Pol B also interacts with the sugar moiety of the incoming ddNTP by way of van der Waals contacts between the ribose ring of the incoming nucleotide and three residues of Pol B . Since the only difference between ribonucleotides and deoxyribonucleotides is a hydroxyl at the 3' carbon of the ribose, these van der Waals interactions may participate in dNTP-NTP differentiation. To further position the incoming ddNTP, there are several additional contacts between the ribose of the ddNTP and Asn279 & Asp276 of Pol B . Numerous other contacts between the phosphates of the ddNTP and Pol B residues are not shown. The ddNTP will eventually be added to the growing DNA strand (primer) .

The 3' OH group of the last sugar poised on the phosphate sugar backbone nucleophilically attacks the phosphate of the incoming ddNTP . In Pol B, the Mg2+ of site B, acting as a Lewis base, activates the 3' OH of the primer, while Asp256 acts as the proton acceptor . In the process, two phosphates from the ddNTP are removed.

The Cis-Peptide
Another interesting feature of the catalytic site is the cis-peptide between Gly274 and Ser274 . Due to the proximity of this cis-peptide with the active site, it is suggested that it plays a role in catalysis. It seems likely that if a cis- to trans- peptide transition occurs, the displacement of helices M and N could occur which in turn could push the polymerase off of the template-primer. Therefore, it is proposed that this cis-peptide facilitates the product-off step of catalysis. Another possibility is that the cis-peptide functions to facilitate the movement of the polymerase along the template during processive polymerization.

Backbone Interactions
There are three different clusters of amino acids of Pol B that interact directly with the DNA backbone. The first cluster is on the amino terminal end of helix G in the fingers subdomain. Here, a cluster of nitrogens are hydrogen bonded to the phosphate backbone of DNA . The second cluster is located in a b-turn which connects b strands 3 and 4 in the palm subdomain . The third DNA-Pol B interaction cluster is located on a loop between b strands 6 and 7 of the thumb subdomain, and backbone phosphates of the DNA .

Base Interactions
Finally, there are three side chains which interact with the bases of the DNA in the minor groove. It has been proposed that their function is to disrupt the water molecule-DNA interactions in the minor groove . If this occurred, the DNA may be conformationally deformed from a B structure to an A-form helix near the active site . 

DNA Binding
There are two structurally homologous DNA binding motifs in Pol B . These consist of helices C and D of the 8 kD domain and helices F and G of the fingers subdomain. These motifs belong to the helix-hairpin-helix (HhH) class of motifs found in many other proteins. Both of these motifs bind metal ions in their hairpin region, as well as interacting with the backbone phosphates of DNA. The HhH motifs may posses lyase ability due to the HhH structural homology between Pol B and the bacterial DNA repair enzyme endonuclease III which catalyzes the cleavage of the DNA phosphodiester backbone. In Pol B, both HhH motifs bind two DNA backbone phosphates.

The HhH in the fingers region associates with DNA from the primer strand, whereas the HhH in the 8 kD domain binds to a gapped-DNA substrate. It is believed that these motifs increase fidelity (nucleotide selectivity) during DNA replication or transcription by forcing the ss DNA template strand to undergo a 90^° bend just before it reaches the active site of the Pol B. This bend likely increases Pol B's sensitivity to mismatched base pairs.

Processivity
There are two metal ion binding sites away from the active site in Pol B. The ions bound at these sites associate with the DNA backbone directly, as well as Pol B. It is believed that the metal ions bound at these sites increase the affinity of Pol B for DNA, as well as allow the enzyme to move smoothly along the DNA.

These sites are contained in the hairpin turn of the HhH motifs . In the metal binding site of the fingers subdomain, the Na⁺ has a square pyramidal coordination geometry with ligands of: threonine 101, valine 103, isoleucine 106, a DNA phosphate oxygen, and a water molecule . One cluster surrounding this Na⁺ is the carbonyl oxygen cluster, located at the carboxyl-terminal end of an a-helix . This cluster contains a negative dipole which stabilizes the positively charged metal ion. The amide nitrogen cluster is located at the amino-terminal end of a different a-helix and possesses a positive helix dipole . This positive charge stabilizes binding to the negatively charged phosphate backbone of the DNA. In particular, four residues are involved, glycine 105, glycine 107, serine 109, and alanine 110 .

These residues are all important for replication, but the metal is essential for processivity. There are two mechanistic explanations for this requirement. First, perhaps the metal ion is bound to Pol B and increases the affinity of the enzyme for DNA while at the same time facilitating movement along the template. However, metal ion bound to Pol B without a DNA substrate has yet to be observed. Therefore, another possible role of metal ions in promoting processivity would be for them to coat the DNA backbone. In this case, Pol B would slide along the DNA on a rail of metal atoms.

Fidelity
As previously discussed, the fidelity exhibited by Pol B may be associated with a 90^° bend of the ss DNA template overhang of the template/primer, causing the last nucleotide of the template to be unstacked before entering the active site. It has been hypothesized that the bending-induced removal of the template nitrogenous base from the stack of bases below it increases fidelity by ensuring that stacking forces between the primer strand stack and the base of an incoming nucleotide do not promote the association of an incorrect base with the template. Once the correct match has been made, the template and new nucleotide are brought back into stacking register with the rest of the helix below them and catalysis can proceed.

Pelletier, H., M. Sawaya, A. Kumar, S. Wilson, and J. Kraut (1994). Structures of Ternary Complexes of Rat DNA Polymerase Beta, a DNA Template-Primer, and ddCTP. Science 264: 1891- 1903.

Pelletier, H., M. Sawaya, W. Wolfle, S. Wilson, and J. Kraut (1996). Crystal Structures of Human DNA Polymerase B Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity. Biochemistry 35: 12742-12761.

Sawaya, M., H. Pelletier, A. Kumar, S. Wilson, and J. Kraut (1994). Crystal Structure of Rat DNA Polymerase B: Evidence for a Common Polymerase Mechanism. Science 264: 1930-1935.