DNA mismatch recognition and binding in human cells has been thought to be mediated by the hMSH2 protein. Here it is shown that the mismatch-binding factor consists of two distinct proteins, the 100-kilodalton hMSH2 and a 160-kilodalton polypeptide, GTBP (for G/T binding protein). Sequence analysis identified GTBP as a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the finding that tumor-derived cell lines devoid of either protein are also devoid of mismatch-binding activity.

GOT mispairs, the sole mismatch type that can arise in "resting" mammalian DNA (through spontaneous hydrolytic deamination of 5-methylcytosine), are corrected in vivo with high efficiency and mostly to a GiC. We identified a protein factor, present in HeLa cell extracts, that binds selectively to DNA substrates containing this mismatch. The partially purified protein was shown by gel-filtration chromatography and UV cross-linking experiments to have an apparent molecular mass of 200 kDa. Its binding to GOT mispairs was not influenced by sequences flanking the mismatch, but methylation of guanines either within the mismatch itself or in its immediate vicinity abolished the formation of the protein-DNA complex. The protein appears to lack both endo-and exonuclease activities and requires neither magnesium nor zinc nor ATP for binding. We discuss the possible role of this protein in a repair pathway, which helps mammalian cells counter the mutagenic effect of the hydrolytic deamination of 5-methylcytosine.

A non-histone protein, NHP1, that binds with high affinity to the estrogen response element (ERE), GGTCAGCGTGACC, has been purified approximately 45,000-fold from HeLa cells by a combination of chromatography on Sephacryl S-300, heparin-Sepharose, Mono Q (FPLC), and sequence-specific oligonucleotide-Sepharose. The native protein has a molecular weight of 170,000 and is composed of two polypeptides of 85 and 75 kDa. The two polypeptides are different as judged by peptide mapping, and only the 85-kDa polypeptide can be cross-linked to the bromodeoxyuridine-substituted synthetic ERE by UV irradiation. The native protein binds to the ERE with an apparent KD of 1 x 10(-11) M and has a pI of 5. The contact points of the protein with individual bases of the ERE have been determined by using partially depurinated and depyrimidinated synthetic oligonucleotides. The strongest contact points of NHP1 with the ERE are 5'AGCG3' in the center of the palindrome and differ from those of the estrogen receptor. NHP1 appears to produce specific nicks around the central CpGs of the ERE, thereby suggesting that it may play a role in active demethylation of mCpGs.

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