Recombination contributes to the structural diversity of HIV, allowing it to evade therapy. We are using segments of the HIV genome to model replication and recombination in vitro. We use cloned reverse transcriptase (RT), and RT mutants to examine the transfer of the growing primer strand from one genome to another, and the remarkably high level of mutation that accompanies this process.

We are also studying the properties of mutants of the RT generated during treatment with anti-AIDS drugs. Using purified proteins, we have reconstituted the reactions required for the synthesis and joining of nascent DNA segments during mammalian DNA replication. This reaction involves priming and synthesis by DNA polymerase alpha, and the action of an unusual nuclease that removes the RNA primers prior to ligation. The nuclease can cut DNA and RNA endonucleolytically, but somehow recognizes only nascent DNA. The secret may lie in the need to recognize a 5' polymer end, and then track to the point of cleavage.

We are also investigating the mechanism by which estrogen induces the expression of specific genes. Interaction of the hormone with a receptor protein in cell nuclei causes the complex to bind estrogen responsive element (ERE) sequences near the promoters of responsive genes. We are examining how variations in the number of adjacent EREs, their spacing, sequence, and sequence context modulate the response of individual genes. Unique effects of the anti-estrogen, anti-tumor drug tamoxifen on this process are also being examined.

Steps of mammalian nascent chromosomal DNA processing:

Muyan M, Yi P, Sathya G, Willmert LJ, Driscoll MD, Hilf R, Bambara RA. Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists. Mol Cell Endocrinol. 182:249-63, 2001.<

Tom S, Henricksen LA, Park MS, Bambara RA. DNA ligase I and proliferating cell nuclear antigen form a functional complex. J Biol Chem. 276:24817-25, 2001.

Archer RH, Wisniewski M, Bambara RA, Demeter LM. The y181c mutant of hiv-1 reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors alters the size distribution of RNAseH cleavages. Biochemistry 40:4087-95, 2001.

Wisniewski M, Balakrishnan M, Palaniappan C, Fay PJ, Bambara RA. Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H. Proc Natl Acad Sci U S A. 97:11978-83, 2000.

Chafin DR, Vitolo JM, Henricksen LA, Bambara RA, Hayes JJ. Human DNA ligase I efficiently seals nicks in nucleosomes. EMBO J. 19:5492-501, 2000.

Wisniewski M, Balakrishnan M, Palaniappan C, Fay PJ, Bambara RA. The sequential mechanism of HIV reverse transcriptase RNase H. J Biol Chem. 275:37664-71, 2000.

Wisniewski M, Balakrishnan M, Palaniappan C, Fay PJ, Bambara RA. Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H. Proc Natl Acad Sci U S A. 97:11978-83, 2000.

Henricksen LA, Tom S, Liu Y, Bambara RA. Inhibition of flap endonuclease 1 by flap secondary structure and relevance to repeat sequence expansion. J Biol Chem. 275:16420-7, 2000.

Tom S, Henricksen LA, Bambara RA. Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1. J Biol Chem. 275:10498-505, 2000.