The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas

The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G₁-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.     

125I]-antigammaglobulin antibodies as universal reagents in radioimmunochemical methods of investigation

A new indirect radioimmunochemical method based on the use of [125I]-anti-IgG-antibodies as universal detecting reagents is proposed. Its first step consists in the antibody binding to the antigen to be analyzed; the second step -- in immunoadsorption of the non-bound antibodies by water-insoluble sorbents prepared by chlorocarbonic acid isobutyl ester copolymerization of antigens with serum albumin or by immobilization of the antigen on Sepharose. The third step is the determination of the amount of sorbent-bound antibodies by means of [125I]-anti-IgG-antibodies. The method proposed was used for quantitative estimation of prolactin, somatotropin, lutropin, BB-isoenzyme of human creatine phosphokinase, testosterone and 5 alpha-dihydroxytestosterone. The method does not employ labelled antigens and is highly sensitive and highly specific.