The production of circulating blood cells from bone marrow stem cells during hematopoiesis is accompanied by overall changes in gene expression which cause production of required functional proteins, such as hemoglobin in erythroid cells, as well as control of cell growth, preventing apoptosis of differentiating cells. Hematopoietic gene regulation is controlled by several specific transcription factors, including the factor Gata-1, which is required for erythrocyte maturation. Based on contacts observed in the NMR structure of the cGata-1 binding domain in complex with DNA, the protein's key DNA interface is interesting in being quite hydrophobic in nature, due to the presence of three leucine side chains protruding toward the DNA. Given the T-rich composition of the GATA DNA binding site, it is possible that thymine's unique 5-methyl group may mediate some of these hydrophobic contacts to increase the stability of binding. The hypothesis that thymine methyl groups are important to the free energy of binding between Gata and DNA is tested by measuring binding of an oligonucleotide substrate in which individual thymine bases are substituted with uracil. To test for any important base-pair specific interactions which may be hydrogen-bonded in character, we have also assayed Gata binding to oligonucleotides with base analogs which cannot make hydrogen bonds. We report that out of the binding site's five thymine methyl groups, only one appeared to make a notable contribution to binding affinity, with removal causing a loss of less than 1 kcal/mol of binding free energy. On the other hand, perturbing the potential hydrogen-bonding surface of the DNAs major groove was found to cause a larger decrease in binding affinity than removal of any of the thymine methyl groups, with a loss of 2-3 kcal/mol of binding free energy. (C) 2004 Elsevier Inc. All rights reserved.