Untangling the Helicobacter pylori genome: Deciphering the significance of zinc finger motif(s) in Topoisomerase I catalysis

Abstract

Helicobacter pylori, a human pathogen dominating gastric microbial population, displays differential gene expression during various stages of stomach colonization. Topoisomerases play a crucial role in maintaining DNA superhelicity and therefore gene expression. H. pylori has only two topoisomerases: DNA gyrase and Topoisomerase I, as opposed to four in most other prokaryotes. The current study focuses on studying the biochemical and mechanistic details of H. pylori Topoisomerase I (HpTopoI) catalysis. Sequence comparison of HpTopoI with EcTopoI shows the presence of four zinc finger motifs (ZFs) at the carboxyl terminal domain (CTD) unlike three in EcTopoI. To understand the role of ZFs in HpTopoI function, the ZFs were sequentially deleted from the carboxyl terminus. It was observed that the third and fourth ZFs are dispensable for HpTopoI function. DNA relaxation activity was hampered considerably when only one zinc finger (ZF1) was present. Deletion of all ZFs, however, drastically reduced DNA binding and abolished DNA relaxation. These results highlight the importance of ZF1 in catalyzing the relaxation of DNA. Furthermore, the annotated active site tyrosine residue in HpTopoI when mutated to phenylalanine retained its DNA relaxation activity. Intriguingly, the CTD HpTopoI alone (all four ZFs) could relax the supercoiled DNA although with a specific activity 8.28 fold less than the WT. Gel filtration chromatography analysis suggests that the CTD occurs as dimer in solution and has an ability to form multimers as shown by glutaraldehyde crosslinking. The amino terminal ‘Toprim’ domain houses an acidic triad DxDxE which co-ordinates a Mg2+ cation that is indispensable for the re-ligation activity during the DNA relaxation process. The deletion of Toprim domain or the triple mutation of the acidic triad residues DxDxE to AxAxE reduced the DNA relaxation activity by 14 fold and 9.5 fold, respectively. HpTopoI has a total of 31 tyrosines. An HpTopoI mutant with all the tyrosines mutated to phenylalanine was over-expressed and purified. This mutant is completely inactive which reinstates the fact that it is indeed tyrosine(s) which bring out the nucleophilic attack on the DNA which is essential for DNA relaxation. A comprehensive analysis of 77 fully sequenced strains of H. pylori revealed the presence of multiple copies of HpTopoI. Out of 77 strains, H. pylori strain XZ274 has emerged as a unique paradigm. This strain was isolated from a gastric cancer patient from Tibet. It has 3 genes annotated as TopoI. This strain lacks a FL copy of HpTopoI. Intriguingly, one of the variant is 317 bp in length encodes for protein with only two zinc fingers. Topoisomerases have been known to interact with several proteins involved in replication, transcription and recombination. In H. pylori, DprA is a crucial protein involved in natural transformation. This study shows that HpDprA stimulates HpTopoI activity at a lower concentration and interacts physically with HpTopoI as demonstrated by SPR and microscale thermophoresis. This indicates a possible role of HpTopoI during the process of natural transformation. Taken together, this study reports the biochemical characterization of HpTopoI and sheds light on the unusual role of zinc finger motifs in enzyme catalysis. Analysis of redundant TopoI copies across several H. pylori strains corroborates with the in vitro results that HpTopoI CTD alone can function as a DNA relaxase