Molecular insights into replication initiation in a multipartite genome harboring bacterium Deinococcus radiodurans

Ganesh K Maurya,Reema Chaudhary,Neha Pandey,Hari S Misra

doi:10.1016/j.jbc.2021.100451

Abstract

Deinococcus radiodurans harbors a multipartite ploid genome system consisting of two chromosomes and two plasmids present in multiple copies. How these discrete genome elements are maintained and inherited is not well understood. PprA, a pleiotropic protein involved in radioresistance, has been characterized for its roles in DNA repair, genome segregation, and cell division in this bacterium. Here, we show that PprA regulates ploidy of chromosome I and II and inhibits the activity of drDnaA, the initiator protein in D. radiodurans. We found that pprA deletion resulted in an increased genomic content and ploidy of both the chromosomal elements. Expression of PprA in trans rescued the phenotypes of the pprA mutant. To understand the molecular mechanism underlying these phenotypes, we characterized drDnaA and drDnaB. As expected for an initiator protein, recombinant drDnaA showed sequence-specific interactions with the putative oriC sequence in chromosome I (oriCI). Both drDnaA and drDnaB showed ATPase activity, also typical of initiator proteins, but only drDnaB exhibited 5′→3′ dsDNA helicase activity in vitro. drDnaA and drDnaB showed homotypic and heterotypic interactions with each other, which were perturbed by PprA. Interestingly, PprA has inhibited the ATPase activity of drDnaA but showed no effect on the activity of drDnaB. Regulation of chromosome copy number and inhibition of the initiator protein functions by PprA strongly suggest that it plays a role as a checkpoint regulator of the DNA replication initiation in D. radiodurans perhaps through its interaction with the replication initiation machinery.

Highlights

The origin of replication in bacterial chromosome is a discrete locus that contains AT-rich conserved DNA motifs and a varying number of 9 mer repeats of nonpalindromic sequences called DnaA boxes
The DNA content and the copy number of genome elements in pprA deletion mutant were compared with the wild-type D. radiodurans
When we checked the copy number of each replicon per cell, we found that the average copy number of chromosome I (Chr I) and chromosome II (Chr II) was 2.5- to 3-fold higher in ΔpprA mutant as compared with the wild type

Summary

Results

The regulatory role of PprA in cell division and genome maintenance has been demonstrated [23, 24, 27, 28]. The DNA content and the copy number of genome elements in pprA deletion mutant were compared with the wild-type D. radiodurans. Further we checked the effect of in trans expression of PprA on the copy number of these genome elements in wild type and ΔpprA mutant. The recombinant drDnaA showed sequence-specific interaction with oriCI as the binding did not change even in the presence of 50-fold excess of cold nonspecific DNA (Fig. 3, A and B). The affinity of drDnaA for a perfect DnaA box (TTATCCACA) was approximately twofold higher (Kd = 2.88 ± 0.28 μM) than the affinity (Kd = 4.21 ± 0.15 μM or 4.36 ± 0.18 μM) for imperfect DnaA boxes (TTTTCCACA or GTATCCACA) (Table 1) These results suggested that drDnaA binds to oriCI in a sequence-specific manner and this interaction is stimulated by ATP. These results suggested that the C-terminal domain IV of drDnaA is

11 Repeats

Discussion

Experimental procedures