Mycobacterial DnaB helicase intein as oxidative stress sensor.

Inteins are widespread self-splicing protein elements emerging as potential post-translational environmental sensors. Here, we describe two inteins within one protein, the Mycobacterium smegmatis replicative helicase DnaB. These inteins, DnaBi1 and DnaBi2, have homology to inteins in pathogens, splice with vastly varied rates, and are differentially responsive to environmental stressors. Whereas DnaBi1 splicing is reversibly inhibited by oxidative and nitrosative insults, DnaBi2 is not. Using a reporter that measures splicing in a native intein-containing organism and western blotting, we show that H2O2 inhibits DnaBi1 splicing in M. smegmatis. Intriguingly, upon oxidation, the catalytic cysteine of DnaBi1 forms an intramolecular disulfide bond. We report a crystal structure of the class 3 DnaBi1 intein at 1.95 Å, supporting our findings and providing insight into this splicing mechanism. We propose that this cysteine toggle allows DnaBi1 to sense stress, pausing replication to maintain genome integrity, and then allowing splicing immediately when permissive conditions return.

Helicases that underpin replication of protein-bound DNA in Escherichia coli.

A pre-requisite for successful cell division in any organism is synthesis of an accurate copy of the genetic information needed for survival. This copying process is a mammoth task, given the amount of DNA that must be duplicated, but potential blocks to replication fork movement also pose a challenge for genome duplication. Damage to the template inhibits the replication machinery but proteins bound to the template such as RNA polymerases also present barriers to replication. This review discusses recent results from Escherichia coli that shed light on the roles of helicases in overcoming protein-DNA barriers to replication and that may illustrate fundamental aspects of how duplication of protein-bound DNA is underpinned in all organisms.

DnaB and DnaI temperature-sensitive mutants of Staphylococcus aureus: evidence for involvement of DnaB and DnaI in synchrony regulation of chromosome replication.

DnaB and DnaI proteins conserved in low-GC content Gram-positive bacteria are apparently involved in helicase loading at the replication initiation site and during the restarting of stalled replication forks. In this study, we found five novel dnaB mutants and three novel dnaI mutants by screening 750 temperature-sensitive Gram-positive Staphylococcus aureus mutants. All of the mutants had a single amino acid substitution in either DnaB or DnaI that controlled temperature-sensitive growth, as confirmed by transduction experiments using phage 80alpha. DNA synthesis as measured by [(3)H]thymine incorporation, origin-to-terminus ratios and flow cytometric analysis revealed that the dnaB and dnaI mutants were unable to initiate DNA replication at restrictive temperatures, which is similar to previous findings in Bacillus subtilis. Furthermore, some of the mutants were found to exhibit asynchrony in the initiation of DNA replication. Also, a fraction of the dnaI mutant cells showed arrested replication, and the dnaI mutant tested was sensitive to mitomycin C, which causes DNA lesions. These results suggest that DnaB and DnaI are required not only for replication initiation and but also for regulation of its synchrony, and they provide support for the involvement of DnaI activity in the restart of arrested replication forks in vivo.