Crystallization and preliminary crystallographic analysis of the complex of the second and third regulatory subunits of human Pol δ

Abstract

Human DNA polymerase (Pol ) consists of four subunits: p125, p50, p66 and p12. A heterodimer containing a His-tagged p50 subunit (p50) and a p50-interacting domain of the p66 subunit (p66 N ) was crystallized. The crystal was in the form of a prism with a rhombic cross-section and belonged to space group P2 1 . The crystal had unit-cell parameters a = 95.13, b = 248.54, c = 103.46 Å , = 106.94 and diffracted to a resolution of 3 Å . Four molecules of p50-p66 N in an asymmetric unit corresponded to a crystal solvent content of 72.2%.

X-ray structure of the complex of regulatory subunits of human DNA polymerase delta

“…Preparations of p50•p66 N samples, crystallization, cryoprotection of crystals, X-ray diffraction data collection and processing are described elsewhere 40. Heavy-atom derivatives were obtained by soaking of crystals in reservoir solutions with dissolved Hg-, Pt-, Au-, Pb- and Sm-containing compounds selected from the Hampton Research heavy atom screens.…”

“…Heavy-atom derivatives were obtained by soaking of crystals in reservoir solutions with dissolved Hg-, Pt-, Au-, Pb- and Sm-containing compounds selected from the Hampton Research heavy atom screens. Cryoprotection of the derivative crystals and diffraction data collection were performed as described for native crystals 40. Complete diffraction data sets were collected and processed using 37 cryoprotected derivative crystals.…”

Functional mapping of the fission yeast DNA polymerase δ B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis

“…A low-resolution structure of the budding yeast Pol ε complex was also obtained by cryo-EM reconstruction [ 9 ]. However, high-resolution structures are now available for the complex of p50 (the second subunit) and the N-terminal 144 amino acids of p66 (the third subunit) of human Pol δ (designated p50•p66 N ) [ 10 , 11 ] and for the complex of the C-terminal domain of budding yeast Pol1 protein, the catalytic subunit of Pol α, and the C-terminal PDE and OB fold domains of the Pol α B-subunit Pol12 [ 12 ].…”

“…In contrast to PCNA and Pol α-primase, Cdc1 binds the N-terminal domain of Cdc27 [ 23 ]. The structure of p50•p66 N complex [ 10 , 11 ] (where p50 and p66 are human orthologues of Cdc1 and Cdc27) now enables the mapping of the interactions within the Cdc1Cdc27 assembly. Figure 1 provides a summary view of the human p50•p66 N complex (Figure 1A ) together with a schematic representation of the domain structure of the two proteins in humans and yeast (Figure 1B ).…”