NMR Structures of a Nonapeptide from DNA Binding Domain of Human Polymerase-α Determined by Iterative Complete-Relaxation-Matrix Approach

Abstract

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from the DNA binding domain of human polymerase-alpha, were determined by complete relaxation matrix analysis of transverse NOE data. The structures exhibit a type III turn with residues KCPC, and the remaining residues exhibit non-ordered structures. The turn was confirmed by alpha, N (i,i+3) connectivity, a low temperature coefficient of NH chemical shift (-3.1 x 10(-3)) of the fourth residue, 3J(NHalpha) coupling constants, and characteristic CD peaks at 228 and 200 nm. Furthermore, phi and psi dihedral angles for the i + 1, and i + 2 residues of the turn are found to be -80 and -41 and -60 and -40 degrees. The first proline residue is trans- while the second exists in both cis- and trans- configurations, with trans- being more than 80% populated. The trans-configuration was established from C5alpha-P6alpha correlation and phi and psi angles of the proline. The five-membered proline ring is in DOWN puckered (C-beta-exo/C-gamma-endo) conformation. The structure of the peptide reveals that the two cysteine thiols are approximately 5 A(o) apart and appropriately positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used anti-cancer drug.

Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases

“…Finally, it was shown that cis-platin binds to thiols much stronger than it binds with guanine [51]. This led to the hypothesis that cis-platin inhibits DNA polymerase-α by binding to the zinc-binding domain, displacing the zinc, and perturbing the structure of the domain [52,53]. It is thought the enzyme recognizes the DNA substrate through the zinc-binding domain, so structural perturbation may interfere with the ability of the enzyme to recognize its substrate.…”

“…We have also shown that cisplatin binds to the Zn-binding domain of DNA polymerase-α approximately 1000 times stronger than binding to guanine [52]. Structural studies of the Zn-binding domain of DNA polymerase-α have shown that divalent Pt (II) binds, to vicinal cysteine residues within the protein [53]. These studies have led to the hypothesis that during cis-platin induced apoptosis, in addition to DNA degradation by DFF, synthesis of DNA may also be inhibited.…”

Post-translational and transcriptional regulation of glycolipid glycosyltransferase genes in apoptotic breast carcinoma cells: VII. Studied by DNA-microarray after treatment with l-PPMP

“…cis-platin is proved to be inhibiting DNA polymerase-alpha [47] and the Helicase III [18,48] in these breast carcinoma cells. It is also established that cis-platin binds to the Zn-binding domain in the active site of DNA polymerase-alpha [49,50]. The exact relation of the inactivation of the replication complex and initiation of apoptotic process is not known [17,48].…”