1. Jose Chang PID: 2932232 January 14, 2014
Ronald E. McNair Post-baccalaureate Achievement Program
Title: Design and engineer a “sequence-specific” DNA topoisomerase to study DNA
topology and DNA topological barriers
Research mentor: Fenfei Leng (Ph.D)
Abstract:
DNA topoisomerases are essential enzymes that convert different topological forms of DNA
during DNA replication, recombination, and transcription. However, most DNA topoisomerases
do not recognize a specific sequence and cannot be used for sequence-specific studies of DNA.
Nevertheless, previous studies showed that vaccinia topoisomerase specifically recognizes 5’-
(C/T)CCTT-3’ and cleaves DNA templates (linear and circular) carrying this pentapyrimidine
sequence. Recent studies, however, showed that using an engineered plasmid mini-circle that
does not contain any 5’-CCCTT-3’ sites (or anything close), vaccinia topoisomerase I still
relaxes the DNA about 10-fold slower than if a 5’-CCCTT-3’ site is present. In this study, we
will design and engineer a “sequence-specific” DNA topoisomerase where a zinc finger DNA-
binding protein (ZFDP) will fuse to the N-terminus of vaccinia topoisomerase. As a start, we will
fuse the ZFDP of the four-finger CCR5 ZFN-R (zinc finger nuclease targeting the CCR5 coding
region) recognizing 5’-AAACTGCAAAAG-3’ to the N-terminus of the vaccinia topoisomerase.
A his-tag will also be added to the N-terminus of the “chimeric” DNA topoisomerase to facilitate
protein purification by Ni-affinity chromatography. Additionally a 21-aa glycine linker
(Gly4Ser3)3 will be inserted between the ZFDP and vaccinia topoisomerase to add flexibility of
the linker region. Plasmid pET30a carrying the coding sequence of the “chimeric” DNA
topoisomerase will be used to transform E. coli strain BL21(DE3). The expression of the
“chimeric” protein will be induced by the addition of IPTG. The hybrid topoisomerase will be
purified by using Ni-affinity chromatography followed by ion-exchange chromatography. After
we purify the “chimeric” DNA topoisomerase, we will test whether it specifically recognizes and
relaxes supercoiled plasmid DNA templates that carry the 5’-AAACTGCAAAAG-3’ sequence.
We will also use it to study DNA topological barriers.