Replication Fork Reactivation in a dnaC2 Mutant at Non-Permissive Temperature in Escherichia coli

Replicative helicases unwind double-stranded DNA in front of the polymerase and ensure the processivity of DNA synthesis. In Escherichia coli, the helicase loader DnaC as well as factors involved in the formation of the open complex during the initiation of replication and primosomal proteins during the reactivation of arrested replication forks are required to recruit and deposit the replicative helicase onto single-stranded DNA prior to the formation of the replisome. dnaC2 is a thermosensitive allele of the gene specifying the helicase loader; at non-permissive temperature replication cannot initiate, but most ongoing rounds of replication continues through to completion (18% of dnaC2 cells fail to complete replication at non-permissive temperature). An assumption, which may be drawn from this observation, is that only a few replication forks are arrested under normal growth conditions. This assumption, however, is at odds with the severe and deleterious phenotypes associated with a null mutant of priA, the gene encoding a helicase implicated in the reactivation of arrested replication forks. We developed an assay that involves an abrupt inactivation of rounds of synchronized replication in a large population of cells, in order to evaluate the ability of dnaC2 cells to reactivate arrested replication forks at non-permissive temperature. We compared the rate at which arrested replication forks accumulated in dnaC2 priA+ and dnaC2 priA2 cells and observed that this rate was lower in dnaC2 priA+ cells. We conclude that while replication cannot initiate in a dnaC2 mutant at non-permissive temperature, a class of arrested replication forks (PriA-dependent and DnaC-independent) are reactivated within these cells

RecG interacts directly with SSB: implications for stalled replication fork regression

RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (−)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression

The involvement of replication in single stranded oligonucleotide-mediated gene repair

Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the ‘annealing-integration’ model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a λ-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased

Evolutionary Convergence and Nitrogen Metabolism in Blattabacterium strain Bge, Primary Endosymbiont of the Cockroach Blattella germanica

Bacterial endosymbionts of insects play a central role in upgrading the diet of their hosts. In certain cases, such as aphids and tsetse flies, endosymbionts complement the metabolic capacity of hosts living on nutrient-deficient diets, while the bacteria harbored by omnivorous carpenter ants are involved in nitrogen recycling. In this study, we describe the genome sequence and inferred metabolism of Blattabacterium strain Bge, the primary Flavobacteria endosymbiont of the omnivorous German cockroach Blattella germanica. Through comparative genomics with other insect endosymbionts and free-living Flavobacteria we reveal that Blattabacterium strain Bge shares the same distribution of functional gene categories only with Blochmannia strains, the primary Gamma-Proteobacteria endosymbiont of carpenter ants. This is a remarkable example of evolutionary convergence during the symbiotic process, involving very distant phylogenetic bacterial taxa within hosts feeding on similar diets. Despite this similarity, different nitrogen economy strategies have emerged in each case. Both bacterial endosymbionts code for urease but display different metabolic functions: Blochmannia strains produce ammonia from dietary urea and then use it as a source of nitrogen, whereas Blattabacterium strain Bge codes for the complete urea cycle that, in combination with urease, produces ammonia as an end product. Not only does the cockroach endosymbiont play an essential role in nutrient supply to the host, but also in the catabolic use of amino acids and nitrogen excretion, as strongly suggested by the stoichiometric analysis of the inferred metabolic network. Here, we explain the metabolic reasons underlying the enigmatic return of cockroaches to the ancestral ammonotelic state

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH–rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites

A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present