Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance inDeinococcus radiodurans

Abstract

31Deinococcus radiodurans, an extremophile, resistant to many abiotic stresses including 32 ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II 33 topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) 34 metabolism was shown in vitro. Here we report that D. radiodurans cells lacking drTopo IB 35 (∆topoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions. 36 The activity of G4 motif containing promoters like mutL and recQ was reduced in the 37 presence of NMM in mutant cells. In mutant cells, the percentage of anucleated cells was 38 more while the ploidy numbers of genome elements were less as compared to wild type. 39 Protein-protein interaction studies showed that drTopo IB interacts with genome segregation 40 and DNA replication initiation (DnaA) proteins. The typical patterns of cellular localization of 41 GFP-PprA were affected in the mutant cells. Microscopic examination of D. radiodurans cells 42 expressing drTopo IB-RFP showed its localization on nucleoid forming a streak parallel to the 43 old division septum and perpendicular to newly formed septum. These results together suggest 44 the role of drTopo IB in genome maintenance in this bacterium. 45 46 Radioresistance; Topoisomerases. 48 49 50 51 52 53 54 55 56 57 58 DNA transaction processes like unwinding, rewinding, movement of proteins on DNA, the 60 formation of higher-order structures during DNA compaction leads to topological 61 entanglements. These topological barriers if unresolved, pose threats to genome stability. 62 DNA topoisomerases are ubiquitous, found in all organisms which resolve the topological 63 constraints in the DNA. Based on the structure and kind of action topoisomerases are 64 classified into type I and type II enzymes. Unlike type II DNA topoisomerases that cleave 65 both the DNA strands and change the linking number in the multiples of two, the type I 66 enzyme reversibly cleaves one strand at a time and the change the linking number in the 67 increment of one. Type I topoisomerases are subdivided into type IA and type IB subfamilies.68 Type IA topoisomerases cleave the DNA strand and make a 5'-phosphotyrosine covenant 69 intermediate with a free 3'-OH strand, while type IB topoisomerases prefer double-stranded 70 DNA, forms a 3'-phosphotyrosine covenant intermediate with a free 5'-OH strand. Type IA 71 enzymes relax only negatively supercoiled DNA, while type IB enzymes relax both negative 72 and positive supercoiled DNA. Both the type I enzymes conserves the energy of the 73 phosphodiester bond in the protein -DNA covalent intermediate, thus overall cleavage and re-74 ligation reactions with DNA strand do not require ATP. Type II topoisomerases are also sub 75 divided into type IIA and type IIB, where type IIA enzymes are present in all domains of life 76 while type IIB enzymes are present in archaea and in some higher plants (Champoux, 2001; 77 Chen et al., 2013). Escherichia coli contains 4 topoisomerases, 2 type I topoisomeras...

FtsK, a DNA motor protein, coordinates the genome segregation and early cell division processes in Deinococcus radiodurans

“…FtsK/SpoIIIE DNA translocases are multidomain and multifunctional proteins that are part of bacterial cell divisome machinery (Mohone and Goley, 2020). In D. radiodurans, our earlier work showed the involvement of ParBs, TopoIB in genome segregation, and FtsZ, FtsA, and DivIVA in cell division (Maurya et al 2019, Modi et al 2014, Chaudhary et al 2019, Kota et al 2021). The co-immunoprecipitation results showed that drFtsK interacts with these proteins indicating its role in both these processes.…”

“…The possible interaction of drFtsK with other deinococcal proteins of cell division like FtsZ, FtsA, and DivIVA, and genome segregation proteins like TopoIB, ParB2, ParB3, and ParB4 was monitored in surrogate E. coli by using the co-immunoprecipitation method. The recombinant pUT18 plasmids were already used in the earlier studies (Maurya et al, 2016, Maurya et al, 2018, Chaudhary et al, 2019, Kota et al, 2021) were transformed in E. coli BL21 (DE3) pLysS strain expressing Histidine tagged FtsK (pETFtsK). E. coli cells co-expressing histidine-tagged FtsK with T18-tagged protein in separate combinations were obtained at the log phase and cell-free extracts were prepared.…”