ASSIGNMENTS OF H-1, N-15, AND C-13 RESONANCES FOR THE BACKBONE AND SIDE-CHAINS OF THE N-TERMINAL DOMAIN OF DNA-POLYMERASE BETA - DETERMINATION OF THE SECONDARY STRUCTURE AND TERTIARY CONTACTS

DNA polymerase beta consists of an N-terminal single-stranded DNA binding domain and a C-terminal catalytic domain separable by mild proteolysis [Kumar et al. (1990) J. Biol. Chem. 265, 2124-2131]. The N-terminal domain participates in template and gapped DNA recognition and contributes significantly to catalysis. The secondary structure and tertiary contacts within the cloned N-terminal domain (residues 2-87) of mammalian DNA polymerase beta have been determined using multidimensional NMR. Assignments of backbone H-1, N-15, and C-13 resonances and side chain]H and C-13 resonances have been obtained from double- and triple-resonance 3D NMR experiments. The C-13-edited TOCSY experiment has allowed nearly complete assignments of H-1 and C-13 resonances within side chains. The C-13-edited NOESY experiment has been used for determination of medium- and long-range NOEs and a determination of tertiary contacts. The N-terminal domain is found to consist of four helices, helix-1 (15-26), helix-2 (36-47), helix-3 (56-61), and helix-4 (69-78), which on the basis of long-range NOEs are tightly packed to form a hydrophobic core. The remainder of the domain consists of two turns (48-51 and 62-65), an Omega-type loop (27-35), and extended structure. The aromatic side chains of Y36, Y39, Y49, and F76 display tertiary contacts indicative of at least partial hydrophobic packing. The S30 and 1-134 residues which cross-link to single-stranded DNA [Prasad et al. (1993) J. Biol. Chem. 268, 15906-15911] are contained within the K27-K35 loop. K72 which is protected from pyridoxal 5'-phosphate modification by dNTP in the intact beta-Pol [Basu et al. (1989) Biochemistry 28, 6305-6309] is found in helix-4. The cross-linking and chemical modification suggest a site of ssDNA template interaction and support a hypothesis that an or-helix (helix-4) similar to the O-helix in the Klenow fragment is important for catalysis by beta-Pol.