Multienzyme complex for metabolic channeling in mammalian DNA replication

阅读量：

作者：

展开

摘要：

In the DNA-synthesizing phase (S phase) of CHEF/18 Chinese hamster embryo fibroblast cells, six enzymes associated with DNA metabolism, including DNA polymerase (deoxynucleoside triphosphate:DNA deoxynucleotidyl-transferase, EC 2.7.7.7), were largely localized in the nuclear region (karyoplasts). By contrast, in quiescent and G1phase cells these enzymatic activities were mainly absent from the nucleus and were recovered in the cytoplasmic portion (cytoplasts). These nuclear (but not cytoplasmic) enzymatic activities cosedimented rapidly on sucrose density gradients. Further, the rapidly sedimenting enzyme activities were unique to cells in S phase. An organized supramolecular structure that allows channeling of metabolites into DNA was demonstrated by kinetics of nucleotide incorporation. ``Permeabilized'' cells selectively channeled incorporation of ribonucleoside diphosphates into DNA in preference to deoxyribonucleoside triphosphates. Deoxyribonucleoside triphosphate incorporation occurred when ribonucleoside-diphosphate reductase (2′-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2′-oxidoreductase, EC 1.17.4.1) activity was abolished by hydroxyurea. Our interpretation is that during DNA replication, the nucleus contains a complex of DNA precursor-synthesizing enzymes juxtaposed with the ``replication apparatus'' comprising DNA polymerase, other enzymes, and structural proteins. Functional integrity of this structure is impaired when one of its essential components is inactivated. We propose the name ``replitase'' for this multienzyme complex for DNA replication and suggest that it incorporates precursors rapidly and efficiently. Possibly its assembly signals the initiation of the S phase of the cell cycle.

展开

关键词：

Biochemistry Replitase Ribonucleoside Diphosphate Reductase Permeabilized Cells Cell Cycle

DOI：

10.1073/pnas.77.6.3312

被引量：

562

年份：

1980