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Human topoisomerase I
introduces transient nicks in duplex DNA to provide swivels that relieve
torsional strain associated with DNA replication, transcription,
recombination and chromatin remodeling.
As one of the DNA strands is cleaved, a tyrosine residue in the enzyme
becomes covalently attached to the 3' phosphate at the site of the nick. In collaboration with Dr. Wim Hol's group,
we recently elucidated the crystal structure of human topoisomerase I in both
noncovalent and covalent complexes with a 22 base pair duplex oligonucleotide
(1).
Current Studies are aimed at understanding (i) the structural basis
for the preference of the enzyme for supercoiled over relaxed DNA, (ii) the
mechanisms of catalysis and relaxation, (iii) the interactions of
topoisomerase I with other proteins in the nucleus, (iv) the conformational
changes of the protein that accompany substrate binding, and (v) the
mechanism of action of the potent anticancer drug camptothecin.
Certain kinds
of DNA damage, the incorporation of base analogs into DNA and treatment of
cells with camptothecin can lead to the covalent trapping of topoisomerase I
on the DNA. Tyrosyl-DNA
phosphodiesterase 1 (Tdp1) has been implicated in the repair of such
complexes by virtue of its ability to hydrolyze a phosphodiester link between
a tyrosine and a DNA 3' phosphate.
Current studies include the purification and biochemical
characterization of a recombinant form of Tdp1. Special emphasis will be on understanding the structure,
substrate specificity, catalytic mechanism, and the in vivo role of the
enzyme. The processing steps that
produce the appropriate substrate in vivo for Tdp1 cleavage are also
being investigated.
Reverse
transcriptase contains an RNase H activity in addition to the well-known DNA
polymerase activity. The RNase H
activity of the enzyme is important for degrading the viral genome RNA after
it has been converted to an RNA-DNA hybrid by the synthesis of the
complementary DNA strand. The RNase H
activity also specifically cleaves the viral RNA in the polypurine tract
(PPT) region to generate the RNA primer used for the initiation of the second
strand of DNA (2). Finally, RNase H is necessary for the
removal of both the tRNA and the PPT RNAs after they have been used to prime
DNA synthesis. Work in the lab is
aimed at understanding the structural basis for these different substrate
specificities. The functional
relationship between the RNase H and DNA polymerase activities of reverse
transcriptase is also being explored.
A thorough understanding of the function of RNase H in reverse
transcription is a prerequisite to the design of inhibitors that can be used
therapeutically.
DNA displacement activity of reverse transcriptase
The generation
of the long terminal repeats that flank the double-stranded DNA product of
reverse transcription requires DNA synthesis with concomitant displacement of
a non-template DNA strand. We have
shown that the polymerase activities of Moloney murine leukemia virus and
HIV-1 reverse transcriptases, unlike most cellular DNA polymerases, are
capable of carrying out extensive displacement synthesis (3).
Currently studies are underway to elucidate the mechanism of
displacement synthesis by HIV-1 reverse transcriptase and to identify the
region of the protein responsible for unpairing the non-template strand as
the polymerase translocates along the DNA.
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Crystal Structure
of human tyrosyl-DNA phosphodiesterase
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