Crystal Structure of Yeast DNA Polymerase ε Catalytic Domain

<div><p>DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2_G:C). Pol2_G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2_T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca²⁺ ions at both positions A and B in the polymerase active site and also observe a Ca²⁺ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2_G:C structure are maintained in the Pol2_T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2_T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.</p></div

Data collection and refinement statistics.

<p>A single crystal was used or solving this structure. Values for outermost shells are given in parentheses</p

Comparison between the Pol2_G:C and Pol2_T:A polymerase and exonuclease active sites.

<p>(a) The nascent base pair binding pocket is shaped by residues Val825 (not shown for clarity), Asn828, Ser829, Ty831 and Gly832 from the fingers domain, and by Tyr645 from the palm domain. Tyr431 approaches the incoming nucleotide from the major groove side. Contacts to the G:C and T:A base pairs are interchangeable in the two structures. (b) The polymerase active site is characterized by acidic residues Asp640 and Asp877. The Pol2_G:C (this work) structure has two Ca²⁺ ions (gray spheres) at positions A and B in the polymerase active site. Pol2_T:A structure was crystallized with one Mg²⁺ ion in the active site. (c) Exonuclease active site in Pol2_GC (left) and Pol2_TA (right). Ca²⁺ ion at position B of Pol2_G:C is shown as gray sphere and is coordinated by Asp290, and a water molecule (red sphere). The atom at position A was modeled as water due to its close proximity to the metal ion at position B. In the Pol2_T:A structure, the exonuclease catalytic residues (Asp290 and Glu292) were mutated to alanines and there are no bound metal ions.</p

Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

<div><p>The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCR<u>A</u>C-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-T<b>CCR</b>A<b>C</b>-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-<b>T</b>CCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.</p></div

Structure of Pol2-DNA-dCTP ternary complex.

<p>Pol2 palm, fingers, thumb, exonuclease and N-terminal domains are shown in cyan, yellow, orange, magenta and blue respectively. DNA is in gray, and the templating G and incoming dCTP are in red. The polymerase (Pol) and exonuclease (Exo) active sites are labeled.</p

Molecular basis for DNA specificity changes at position 2, 3, 4, and 6.

<p>Contacts in the structure are shown on the left and the specificity changes are modeled on the right. At position 2, mutation of Lys645 to Met645 converts DNA specificity from C:G to A:T; at position 3, mutation of Glu751 and Asn773 to Arg751 and Asp773 converts DNA specificity from C:G to G:C; at position 4, mutation of Arg810 and Ala774 to Ser810 and Lys774 converts DNA specificity from G:C to C:G; at position 6, mutation of Glu806 and Arg808 to Lys806 and Asp808 converts DNA specificity from C:G to G:C.</p

Crystallographic parameters and refinement statistics.

<p>Crystallographic parameters and refinement statistics.</p

Change in specificity at position 2.

<p>(A) Restriction fragment digestion patterns of lambda, PhiX174, and pBR322 DNAs with wt = wild type MmeI, which cuts at TCCRAC20/18; A = MmeI Lys₆₄₅Met mutant, which cuts at TACRAC20/18; R = MmeI Tyr₆₄₂Lys, Lys₆₄₅Met double mutant, which cuts at TRCRAC20/18; M = size standard, lambda-HindIII digest plus PhiX174-HaeIII digest. (B) Cut site determination for MmeI K₆₄₅M mutant showing cutting at TACRAC20/18. Run-off Sanger sequencing of pUC19 DNA (TACRAC site at 376 to 381), priming from both sides (5' and 3') of the TACRAC recognition site and point of DNA cleavage. (C) Cut site determination for MmeI Y₆₄₂K, K₆₄₅M double mutant showing cutting at both TACRAC20/18 (top panel, pUC19 site at 376 to 381) and TGCRAC (bottom panel, pUC19 site at 1842 to 1847). Run-off Sanger sequencing of the cleaved pUC19 DNA, showing priming from 5' to the TACRAC or TGCRAC recognition site (bottom strand cleavage shown).</p

Interactions with Sinefungin.

<p>The SAM analog Sinefungin is tightly bound within the MTase domain via extensive hydrogen bonding (dashed lines) and hydrophobic contacts.</p

Molecular basis for DNA recognition.

<p>The first base pair (T:A) is specified by hydrophobic contacts with Phe737, Tyr 738, and Ala723 of the TRD; the second base pair (C:G) makes contacts with Lys645 and Tyr642 of the TRD and Lys487 and Ser488 of the MTase domain; the third base pair (C:G) is specified by Glu751 and Asn773 of the TRD; the fourth base pair (R:Y) makes contacts with Arg810 of the TRD and Lys489 of the MTase domain; at the fifth base pair (A:T), the thymine opposite the adenine to be methylated is specified by hydrophobic contacts with Thr752 and Leu805 of the TRD; the sixth base pair (C:G) is specified by Glu806 and Arg808 of the TRD. H-bonds are depicted by dashed lines and distances in Angstroms.</p