Hmo1, an HMG-box protein,belongs to the yeast ribosomal DNA transcription system

Hmo1 is one of seven HMG-box proteins of Saccharo myces cerevisiae. Null mutants have a limited effect on growth. Hmo1 overexpression suppresses rpa49-Delta mutants lacking Rpa49, a non-essential but conserved subunit of RNA polymerase I corresponding to the animal RNA polymerase I factor PAF53. This overexpression strongly increases de novo rRNA synthesis. rpa49-Delta hmo1-Delta double mutants are lethal, and this lethality is bypassed when RNA polymerase II synthesizes rRNA. Hmo1 co-localizes with Fob1, a known rDNA-binding protein, defining a narrow territory adjacent to the nucleoplasm that could delineate the rDNA nucleolar domain. These data identify Hmo1 as a genuine RNA polymerase I factor acting synergistically with Rpa49. As an HMG-box protein, Hmo1 is remotely related to animal UBF factors. hmo1-Delta and rpa49-Delta are lethal with top3-Delta DNA topoisomerase (type I) mutants and are suppressed in mutants lacking the Sgs1 DNA helicase. They are not affected by top1-Delta defective in Top1, the other eukaryotic type I topoisomerase. Conversely, rpa34-Delta mutants lacking Rpa34, a non-essential subunit associated with Rpa49, are lethal in top1-Delta but not in top3-Delta.     

N-terminal region of the large subunit of Leishmania donovani bisubunit topoisomerase I is involved in DNA relaxation and interaction with the smaller subunit

Leishmania donovani topoisomerase I is an unusual bisubunit enzyme. We have demonstrated earlier that the large and small subunit could be reconstituted in vitro to show topoisomerase I activity. We extend our biochemical study to evaluate the role of the large subunit in topoisomerase activity. The large subunit (LdTOP1L) shows a substantial degree of homology with the core DNA binding domain of the topoisomerase IB family. Two N-terminal truncation constructs, LdTOP1Delta39L (lacking amino acids 1-39) and LdTOP1Delta99L (lacking amino acids 1-99) of the large subunit were generated and mixed with intact small subunit (LdTOP1S). Our observations reveal that residues within amino acids 1-39 of the large subunit have significant roles in modulating topoisomerase I activity (i.e. in vitro DNA relaxation, camptothecin sensitivity, cleavage activity, and DNA binding affinity). Interestingly, the mutant LdTOP1Delta99LS was unable to show topoisomerase I activity. Investigation of the loss of activity indicates that LdTOP1Delta99L was unable to pull down glutathione S-transferase-LdTOP1S in an Ni(2+)-nitrilotriacetic acid co-immobilization experiment. For further analysis, we co-expressed LdTOP1L and LdTOP1S in Escherichia coli BL21(DE3)pLysS cells. The lysate shows topoisomerase I activity. Immunoprecipitation revealed that LdTOP1L could interact with LdTOP1S, indicating the subunit interaction in bacterial cells, whereas immunoprecipitation of bacterial lysate co-expressing LdTOP1Delta99L and LdTOP1S reveals that LdTOP1Delta99L was significantly deficient at interacting with LdTOP1S to reconstitute topoisomerase I activity. This study demonstrates that heterodimerization between the large and small subunits of the bisubunit enzyme appears to be an absolute requirement for topoisomerase activity. The residue within amino acids 1-39 from the N-terminal end of the large subunit regulates DNA topology during relaxation by controlling noncovalent DNA binding or by coordinating DNA contacts by other parts of the enzyme.