Selective interaction of 5'-bromodeoxyuridine substituted DNA with different chromosomal proteins.
Chromosomal proteins selectively interact with 5'-bromodeoxyuridine (BrdUrd) substituted DNA relative to unsubstituted DNA. The relative affinities of chromosomal proteins for BrdUrd-DNA and unsubstituted DNA were measured by both thermal chromatography on hydroxylapatite and selective retention on nitrocellulose filters. Certain chromosomal proteins have a high affinity for hydroxylapatite; thus, during thermal chromatography of chromatin, the single-stranded DNA component percolates across a bed of adsorbed proteins as it elutes. We have measured the relative affinities of Brd-Urd-DNA and normal DNA for chromosomal proteins by chromatographing appropriate mixtures on hydroxylapatite. The results show that, under these conditions, the histone components, rather than the nonhistone chromatin proteins, retard the BrdUrd-substituted DNA. In addition, the individual histones vary in the degree of their affinity for BrdUrd-DNA in the order H3 greater than H4 greater than H2A greater than H2B greater than H1. We have used the property that protein-DNA complexes have a preferential affinity for nitrocellulose filters over naked DNA to measure the selective binding of BrdUrd-DNA and unsubstituted DNA's to both histone and nonhistone chromosomal proteins at low temperatures. The histones selectively retained BrdUrd-DNA on filters in the order H4 greater than H2A greater than H3 greater than H2B greater than H1. Using this assay, the nonhistones displayed greater selectivity toward BrdUrd-DNA than the histone fraction. We interpret these results to mean BrdUrd-containing DNA has a specific affinity for certain chromosomal proteins with BrdUrd-DNA may be the basis for selective inhibition of cytodifferentiation by the thymidine analogue, BrdUrd.
D001665:Binding Sites; D001667:Binding, Competitive; D001973:Bromodeoxyuridine / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004247:DNA / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D006657:Histones; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms; D009698:Nucleoproteins; D011485:Protein Binding; D013696:Temperature
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004273:DNA, Neoplasm; D006657:Histones; D009698:Nucleoproteins; D004247:DNA; D001973:Bromodeoxyuridine