alexa The domain organization of human topoisomerase I.
Microbiology

Microbiology

Journal of Antivirals & Antiretrovirals

Author(s): Stewart L

Abstract Share this page

Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH2-terminal domain (approximately 24 kDa), which is known to be dispensable for activity, (ii) the core domain (approximately 54 kDa), (iii) a linker region (approximately 3 kDa), and (iv) the COOH-terminal domain (approximately 10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH2-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH2-terminal domain. When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive charge of the linker domain suggests that at a certain point in catalysis the linker may bind directly to DNA. Further, we show that limited subtilisin cleavage can generate a mixture of 60-kDa core and approximately 10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH2-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies.

This article was published in J Biol Chem and referenced in Journal of Antivirals & Antiretrovirals

Relevant Expert PPTs

Relevant Speaker PPTs

Recommended Conferences

Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords