Carboxyl Terminal Truncations of the Phage Mu Transposase

The Phage Mu transposase (MuA) is a 663 amino acid momomeric multidomain protein. A tetramer of MuA catalyzes the replicative transposition of Phage Mu. MuA pairs the ends of the Mu genome, cleaves the donor DNA and subsequently joins the Mu DNA to the target DNA in a concerted cleavage and joining reaction known as strand transfer. A series of truncations deleting 101, 136 and 173 amino acids from the carboxyl terminal end of MuA were constructed, purified and analyzed. As expected the truncation proteins were inactive when assayed individually under standard in vitro transposition reaction conditions. In the presence of either wild type MuA, or a catalytic site point mutant, MuA D269N/E392Q, the truncation proteins were able to assemble into mixed tetramers that displayed partial catalytic activity. These mixed tetramers were capable of carrying out donor cleavage, and under relaxed reaction conditions, strand transfer. The data suggests that some, but not all, of the multimerization determinants lie in the deleted region of the protein and that the deleted regions also contain a putative nonspecific DNA binding region and catalytic site responsible for donor cleavage. Thesis Supervisor: Tania A. Baker Title: Assistant Professor of Biology and Assistant Investigator, Howard Hughes Medical Institute For My Dearest Friends and Family ACKNOWLEDGEMENTS I would like to thank Tania A. Baker for her unyielding support, without which this work would have never come to pass. Her discussions and comments have been invaluable font of knowledge and intuition and have guided me through my growth as a scientist. I wish to thank the members of the lab for their kindness and support, especially Elena Krementsova for being a jovial baymate and filling my days with mirth, Igor Levchenko for showing unbelieveable drive and of course those crazy skiing trips and Mike Yamauchi for always having ten minutes to spare. I could not have survived MIT without the help and support of my dearest friends and family, especially Kobi, Jan, Al and JoJo, who have stood by me through fair and foul wind alike. A special thanks goes out to a real New Yorker's New Yorker, my good friend Ann-Marie White. Through crosswords, coffee, love, and life she has always been a beacon of civility, and silliness. She came to my rescue two days before the deadline, after a crashed disk, and in the middle of utter and total panic. Above all I wish to thank Laura Beth Willis, for having the strength to be true to herself. Thank you Laura, for sneaking.