1. Volume 25, Issue 4, Pages 601-614 (February 2007)
Human DNA Polymerase κ Encircles DNA: Implications for Mismatch Extension and Lesion Bypass 
Samer Lone, Sharon A. Townson, Sacha N. Uljon, Robert E. Johnson, Amrita Brahma, Deepak T. Nair, Satya Prakash, Louise Prakash, Aneel K. Aggarwal 
Molecular Cell 
Volume 25, Issue 4, Pages (February 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Requirement of N Terminus for Pol κ Function
(A) Schematic alignment of human Pol κ with E. coli DinB (Pol IV) and S. solfataricus Dpo4. Regions corresponding to motifs I–V, common to all Y family polymerases, are shown in different colors. Regions that form the architectural palm, fingers, and thumb domains are shown in red, yellow, and orange, respectively. The x, y, and z motifs in Pol κ are shared among all members of the DinB subfamily. The N-terminal region of ∼75 residues is present in Pol κ but not in DinB or Dpo4.
(B) DNA synthesis from matched and mismatched primer termini opposite from template G. Full-length Pol κ1–870 (0.5 nM) or truncated Pol κ1–526 (0.5 nM), Pol κ19–526 (0.5 nM), or Pol κ68–526 (50 nM) were incubated with 10 nM DNA substrate for 5 min at 37°C under the standard polymerase reaction conditions that contained 25 μM of each of the four dNTPs. In the DNA sequence shown on top, N in the primer refers to any of the four dNTPs.
(C) DNA synthesis from an A paired with an undamaged T or the 3′T of a cis-syn T-T dimer (CPD). Reaction conditions were the same as in (B). A portion of the DNA substrate is shown on top, and the identities of the nucleotide in the template and the primer at the template-primer junction are indicated below each lane.
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3. Figure 2 Pol κ Encircles DNA
(A) Ribbon diagram representing the overall structure of the ternary complex. The palm, fingers, and thumb domains and the PAD are shown in light blue, yellow, orange, and green, respectively. The N-clasp subdomain, unique to Pol κ, is highlighted in dark blue. DNA is represented in gray; template dA and incoming dTTP are in red, and a putative Mg2+ ion is in blue.
(B) A view of the ternary complex looking down the DNA helix to show encirclement of the DNA by the N-clasp.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Conformational Changes in Pol κ and Its Relation to Other Y Family DNA Polymerases
(A) Conformational changes in Pol κ upon complex formation. On binding DNA and incoming dNTP, the PAD domain swings ∼50 Å toward the major groove of the DNA. In addition, the N-clasp appears to undergo a disorder-to-order transition—locking Pol κ around the DNA.
(B) Surface diagrams comparing Pol κ to the structures of several Y family polymerases. The colors of the domains coincide with those used in Figure 2, with the N-digit from Rev1 also highlighted in dark blue.
(C) The secondary structure and domain topology of Pol κ.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 The Pol κ Active-Site Cleft
(A) A close-up view of the Pol κ active-site region. The fingers, palm, and thumb domains and the PAD are shown in yellow, light blue, orange, and green, respectively. The N-clasp is colored dark blue, with the U-shaped tether omitted for clarity. The DNA is colored gray, the template dA and incoming dTTP are in red, and the putative Mg2+ ion is shown in blue. Highlighted and labeled are the catalytic residues (D107, D198, and E199) and the residues apposed close to incoming dTTP (F111, Y112, T138, Y141, R144, and K328), template dA (M135, A151, K461, F465, and R507), and the first unpaired template dC (F49, S134, P153, and F155).
(B) Close-up view of the nascent base pair fitting against the Pol κ molecular surface. Several residues apposed to the templating dA and unpaired dC base are shown in gray and labeled on the molecular surface.
(C) Close-up view of metal ion coordination in the active site. On the left is shown a simulated annealing Fo − Fc omit map (contoured at 3.0 σ; 3.05 Å resolution), showing a single Mg2+ ion in the active site. Highlighted are the catalytic residues (D107, D198, and E199) and M108. The incoming dTTP is shown in red, and the Mg2+ ion is shown as a blue sphere. On the right, the Mg2+ ion coordination geometry is shown in more detail along with the ligation distances (in Å units). Pink star marks the site of putative 3′OH.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 DNA-Binding Affinities of N-Terminally Deleted Pol κ Proteins
(A) Schematic representation of N-terminal deletions generated in Pol κ. Amino acid residues contained in each protein are indicated on the left. Dissociation constant (KD) for each construct is indicated on the right.
(B) Binding curves for the various N-terminally deleted Pol κ constructs. The fraction of DNA bound is plotted versus Pol κ concentration in order to determine the dissociation constants. Filled circles are for Pol κ1–526, open circles for Pol κ19–526, filled-in triangles for Pol κ37–526, open triangles for Pol κ47–526, and filled-in squares for Pol κ68–526.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Modeling of DNA Lesions
(A) Model of a cis-syn cyclobutane T-T dimer. A cis-syn T-T dimer (red) was modeled in the active sites of Pol κ and Pol η with the 3′T at the templating position. Coordinates for the T-T dimer were obtained from the structure of Dpo4 complexed with a cys-syn T-T dimer (Ling et al., 2003). The yeast apo Pol η structure was superimposed onto that of the Pol κ-DNA complex via the palm domain. The fingers domain and N-clasp are highlighted in yellow and dark blue, respectively, with residues apposed to the T-T dimer colored in green. The rest of the template and primer strands are shown in gray, and the incoming dNTP is omitted. In Pol κ, the N-clasp and the fingers domain are in close proximity to the T-T dimer and the 5′T collides with F49 and M135. In contrast, Pol η can readily accommodate the 5′T of the T-T dimer because it lacks an equivalent N-clasp, and the fingers are positioned farther away from the templating strand.
(B) Model of an HNE-dG adduct. An HNE-dG minor groove adduct (cyan) was modeled in the active sites of Pol κ and Pol ι at the postinsertion T1 position. The HNE-dG adduct is shown in the ring-open form with the active sites of Pol κ and Pol ι displayed as surface representations. Flanking DNA bases are omitted for clarity. Pol κ is able to extend past the HNE-dG adduct at the T1 position, whereas the same lesion in Pol ι blocks replication. From the model, the ring-open form of the HNE-dG adduct fits into the minor groove and is unobstructed by Pol κ; the closest Pol κ residues (F171, R175, and L197) are highlighted in white. In contrast, the minor groove is less accessible with Pol ι and the long chain of the HNE-dG adduct clashes with residues Y39 and F125 on the Pol ι molecular surface.
Molecular Cell  , DOI: ( /j.molcel )
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