Purification and nucleic acid binding properties of a fragment of type C1/C2 heterogeneous nuclear ribonucleoprotein from thymic nuclear extracts.

A single-strand nucleic acid binding protein (C/F) that has an apparent molecular weight of 12,000 on SDS-polyacrylamide gel electrophoresis and that was originally thought to be the 12-kDa alpha-subunit of the AB form of terminal deoxynucleotidyl transferase (TdT) from calf thymus has been purified and identified as a fragment of the type C1/C2 hnRNP proteins. On the basis of NH2-terminal sequencing and mass spectrometric analysis, C/F contains approximately 94 residues and spans from residue 9 to approximately residue 102 in the type C1/C2 hnRNP proteins. C/F is presumably produced in vitro via limited proteolysis of the type C1/C2 hnRNP proteins following cell disruption. Since C/F corresponds almost exactly to the approximately 90-residue conserved ribonucleoprotein binding domain (RBD) that is shared by many eukaryotic RNA binding proteins, it provided an opportunity to better characterize the domain structure of the type C1/C2 hnRNP proteins and to compare the nucleic acid binding properties of the type C1/C2 and A1 [see Shamoo et al. (1994) Biochemistry, preceding paper in this issue] RNA binding domains. Like the type A1 RBD, the type C1/C2 RBD has an apparent occluded site size of 6-7 nucleotides. The type C1/C2 RBD binds non-cooperatively to homopolynucleotides and has preferential affinity for RNA and for single as opposed to double-stranded nucleic acids. The type C1/C2 RBD has about a 100-fold higher affinity than the type A1 RBD does for RNA and some of this increased affinity results from additional ionic interactions. The latter account for approximately 50% of the free energy of binding of the type C1/C2 RBD. While the type C1/C2 hnRNP proteins exist in vivo as a very tight tetramer with the structure (C1)3C2 [Barnett et al. (1989) Mol. Cell. Biol. 9, 492-498], the isolated type C1/C2 RBD is a monomer. Hence, the determinants for tetramerization appear to lie outside the type C1/C2 RBD. Phenylalanine 19 was identified as the only point of photochemical cross-linking of the type C1/C2 RBD to [d(T)]8. This residue corresponds to the major site of cross-linking of the A1 RBD to [d(T)]8 [Merrill, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H., & Williams, K. R. (1988) J. Biol. Chem. 263, 3307-3313].(ABSTRACT TRUNCATED AT 400 WORDS)

Thymic nuclear matrix associated activity is not V(D)J recombinase.

It was previously reported that nuclear matrix isolated from young rat thymus contained an activity that supported V(D)J recombination at a high efficiency (Dave et al., BIOCHEMISTRY 30: 4763-4767, 1991). A similar type of activity is also detected in the matrix prepared from fetal calf thymus. However, restriction enzyme mapping analyses of the recombined product clearly suggest that the double antibiotic resistance exhibited by the matrix treated plasmid substrate is not a consequence of V(D)J signal sequence recombination.

Terminal deoxynucleotidyltransferase containing megadalton complex from young rat thymus nuclei: identification and characterization.

Nuclear matrix prepared from 2-3 week old rat thymuses contains tightly bound TdT activity which has been quantitatively solubilized with nonionic detergent and sonication. TdT is contained in a discrete complex with a sedimentation value of 23 S. The complex is retained on an anti-TdT antibody column and contains DNA ligase and 3'-5' exonuclease activities as well as DNA and several other proteins but is devoid of replicative DNA polymerases. Such a type of multienzyme complex is absent from the nuclear extracts of thymus prepared from older rats and also from liver and spleen extracts of young and old rats.

Inhibition of terminal deoxynucleotidyl transferase by adenine dinucleotides. Unique inhibitory action of Ap5A.

Terminal deoxynucleotidyltransferase (TdT) exhibits strong sensitivity to ATP and its dinucleotide analogues, Ap2A, Ap3A, Ap4A, Ap5A and Ap6A. Similar to ATP, all of the dinucleotides appear to be competitive inhibitors of TdT catalysis with respect to substrate deoxynucleoside triphosphates and effectively block the UV-mediated substrate cross-linking to TdT. Among the various dinucleotides, Ap5A and Ap6A (diadenosine 5'-5' penta- and hexaphosphate, respectively) are significantly more effective than dinucleotides containing 2, 3 or 4 phosphate backbones. Furthermore, Ap5A is found to be the only dinucleotide which has reactivity at both substrate- and primer-binding domains in TdT.