A Replication Terminus Located at or Near a Replication Checkpoint of Bacillus subtilis Functions Independently of Stringent Control

Ashish Gautam,Deepak Bastia

doi:10.1074/jbc.m009538200

Abstract

We have examined a replication terminus (psiL1) located on the left arm of the chromosome of Bacillus subtilis and within the yxcC gene and at or near the left replication checkpoint that is activated under stringent conditions. The psiL1 sequence appears to bind to two dimers of the replication terminator protein (RTP) rather weakly and seems to possess overlapping core and auxiliary sites that have some sequence similarities with normal Ter sites. Surprisingly, the asymmetrical, isolated psiL1 site arrested replication forks in vivo in both orientations and independent of stringent control. In vitro, the sequence arrested DnaB helicase in both orientations, albeit more weakly than the normal Ter1 terminus. The key points of mechanistic interest that emerge from the present work are: (i) strong binding of a Ter (psiL1) sequence to RTP did not appear to be essential for fork arrest and (ii) polarity of fork arrest could not be correlated in this case with just symmetrical protein-DNA interaction at the core and auxiliary sites of psiL1. On the basis of the result it would appear that the weak RTP-L1Ter interaction cannot by itself account for fork arrest, thus suggesting a role for DnaB-RTP interaction.

Highlights

Inhibition of protein synthesis by amino acid analogs, such as arginine hydroxamate causes intracellular accumulation of uncharged tRNA, that in turn activates the relA gene
The key points of mechanistic interest that emerge from the present work are: (i) strong binding of a Ter (␺L1) sequence to replication terminator protein (RTP) did not appear to be essential for fork arrest and (ii) polarity of fork arrest could not be correlated in this case with just symmetrical protein-DNA interaction at the core and auxiliary sites of ␺L1
In B. subtilis, induction of stringent response by addition of arginine hydroxamate causes replication forks initiated from oriC to be arrested at check points, that we have labeled as ␺L and ␺R (Fig. 1), that are located ϳ200 kilobases on either sides of oriC [4, 5]

Summary

Introduction

Inhibition of protein synthesis by amino acid analogs, such as arginine hydroxamate causes intracellular accumulation of uncharged tRNA, that in turn activates the relA gene. In B. subtilis, induction of stringent response by addition of arginine hydroxamate causes replication forks initiated from oriC to be arrested at check points, that we have labeled as ␺L and ␺R (Fig. 1), that are located ϳ200 kilobases on either sides of oriC [4, 5]. Arrest at both of the checkpoints requires the replication terminator protein (RTP), suggesting the existence of Ter-like sites about the checkpoints. Checkpoint Terminus terminator-RTP complex still showed appreciable replication arrest in vivo tends to support the conclusion that strong protein-DNA interaction by itself is not obligatory for replication fork arrest at the Ter sites of B. subtilis

Methods

Results

Conclusion