Formation of highly stable complexes between 5-azacytosine-substituted DNA and specific non-histone nuclear proteins. Implications for 5-azacytidine-mediated effects on DNA methylation and gene expression.
Incubation of 5-azacytosine-substituted DNA ([5-aza-C]DNA) with nuclear proteins leads to the formation of highly stable DNA . protein complexes which remain intact in the presence of 1 M NaCl and/or 0.6% Sarkosyl. The proteins involved in binding double-stranded [5-aza-C]DNA in these stable complexes comprise a specific subset of non-histone nuclear proteins that includes DNA methyltransferase. Complex formation does not require S-adenosylmethionine and does not involve covalent linkage of protein to DNA or modification of 5-azacytosine residues. Non-histone nuclear proteins do not form complexes with double-stranded unsubstituted DNA that are resistant to dissociation with NaCl and Sarkosyl but are capable of forming such complexes with single-stranded DNA regardless of whether it contains 5-azacytosine residues or not. However, it can be demonstrated 1) that single-stranded regions do not account for stable binding of proteins to native [5-aza-C]DNA and 2) that many nuclear proteins which form stable complexes with single-stranded DNA are incapable of forming such complexes with double-stranded [5-aza-C]DNA. Synthesis of [5-aza-C]DNA by cells growing in the presence of either 5-azacytidine or 5-aza-2'-deoxycytidine leads to rapid loss of extractable DNA methyltransferase (Creusot, F., Acs, G., and Christman, J.K. (1982) J. Biol. Chem. 257, 2041-2048). Analogous depletion of non-histone nuclear proteins capable of forming stable complexes with [5-aza-C]DNA in vitro is observed, suggesting that the same proteins can form highly stable complexes with [5-aza-C]DNA in vitro and in vivo. Formation of stable complexes between non-histone nuclear proteins and [5-aza-C]DNA could potentially affect not only the activity of DNA methyltransferase but the action of other regulatory proteins or enzymes that interact with DNA. Such interactions could explain effects of 5-azacytidine on gene expression that cannot be directly linked to loss of methyl groups from DNA.
Christman
J K
JK
Schneiderman
N
N
Acs
G
G
D000818:Animals; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D003596:Cytosine / Q000031:analogs & derivatives*; D004247:DNA / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D005786:Gene Expression Regulation; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D015719:Single-Strand Specific DNA and RNA Endonucleases; D012965:Sodium Chloride / Q000494:pharmacology
D002868:Chromosomal Proteins, Non-Histone; C039854:5-azacytosine; D012965:Sodium Chloride; C025231:sarkosyl; D003596:Cytosine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D012521:Sarcosine