Kenneth N. Kreuzer

The importance of repairing stalled replication forks

The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.

Formation and resolution of DNA catenanes by DNA gyrase

We have discovered that DNA gyrase interlocks duplex DNA circles to form catenanes and resolves catenanes into component monomers. The reactions were inhibited by novobiocin and oxolinic acid and required ATP, Mg++ and spermidine. DNA sequence homology is not involved in catenation, since hybrid catenanes were formed efficiently between supercoiled phi X174 and Col E1 DNA. Strikingly different results were obtained with native and relaxed Col E1 DNA substrates. Up to 50-60% of input native DNA was converted into oligomeric catenanes, predominantly dimers and trimers. Relaxed substrates were instead converted into vast interlocked networks and were occasionally knotted. Optimal catenation occurred only in the narrow range of 20-35 mM KCl; increased ionic strength blocked catenation completely but activated the back reaction of decatenation. Gyrase resolved both the oligomeric catenanes and interlocked networks it produced, as well as naturally occurring catenanes. These results imply that the mechanism of gyrase involves a transient double-strand break and passage of a DNA segment through the resulting gap. Gyrase is representative of a general class of enzymes, found in both procaryotic and eucaryotic cells, that facilitate diffusion of duplex DNA segments through each other and may thereby solve topological problems arising from the replication, recombination and condensation of DNA.

Recombination-dependent DNA replication in phage T4

Studies in the 1960s implied that bacteriophage T4 tightly couples DNA replication to genetic recombination. This contradicted the prevailing wisdom of the time, which staunchly supported recombination as a simple cut-and-paste process. More-recent investigations have shown how recombination triggers DNA synthesis and why the coupling of these two processes is important. Results from T4 were instrumental in our understanding of many important replication and recombination proteins, including the newly recognized replication/recombination mediator proteins. Recombination-dependent DNA replication is crucial to the T4 life cycle as it is the major mode of DNA replication and is also central to the repair of DNA breaks and other damage.

Escherichia coli phage T4 topoisomerase

Publisher Summary Topoisomerases are enzymes that alter DNA topology by changing the linking number of circular duplex DNA molecules and by interconverting topologically knotted or catenated DNA forms. The so-called type II topoisomerases act by a mechanism involving the passage of a duplex segment of DNA through a transient double-strand break in another segment of DNA. A novel ATP-dependent type II topoisomerase with DNA-dependent ATPase activity is isolated from extracts of bacteriophage T4-infected E. coli cells. It has a high specific activity for topoisomerization reactions and can be easily purified to near homogeneity in milligram amounts. This chapter explains the interaction of T4 topoisomerase with DNA in the origin region of the T4 chromosome and attempts to reconstitute the initiation reaction in vitro in order to test this and other models for the involvement of the enzyme in the DNA replication process.

Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins

Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker ‘A’ motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW‐K141R, inhibits replication from a T4 origin at which persistent RNA–DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double‐strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA–DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW‐K141R fusion, displays ssDNA‐dependent ATPase activity and is able to unwind a branched DNA substrate.

Isolation of SOS Constitutive Mutants of Escherichia coli

The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.

5-Azacytidine–Induced Methyltransferase-DNA Adducts Block DNA Replication In vivo

5-Azacytidine (aza-C) and its derivatives are cytidine analogues used for leukemia chemotherapy. The primary effect of aza-C is the prohibition of cytosine methylation, which results in covalent methyltransferase-DNA (MTase-DNA) adducts at cytosine methylation sites. These adducts have been suggested to cause chromosomal rearrangements and contribute to cytotoxicity, but the detailed mechanisms have not been elucidated. We used two-dimensional agarose gel electrophoresis and electron microscopy to analyze plasmid pBR322 replication dynamics in Escherichia coli cells grown in the presence of aza-C. Two-dimensional gel analysis revealed the accumulation of specific bubble and Y molecules, dependent on overproduction of the cytosine MTase EcoRII (M.EcoRII) and treatment with aza-C. Furthermore, a point mutation that eliminates a particular EcoRII methylation site resulted in disappearance of the corresponding bubble and Y molecules. These results imply that aza-C-induced MTase-DNA adducts block DNA replication in vivo. RecA-dependent X structures were also observed after aza-C treatment. These molecules may be generated from blocked forks by recombinational repair and/or replication fork regression. In addition, electron microscopy analysis revealed both bubbles and rolling circles (RC) after aza-C treatment. These results suggest that replication can switch from theta to RC mode after a replication fork is stalled by an MTase-DNA adduct. The simplest model for the conversion of theta to RC mode is that the blocked replication fork is cleaved by a branch-specific endonuclease. Such replication-dependent DNA breaks may represent an important pathway that contributes to genome rearrangement and/or cytotoxicity.

Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo.

Antibacterial quinolones inhibit type II DNA topoisomerases by stabilizing covalent topoisomerase‐DNA cleavage complexes, which are apparently transformed into double‐stranded breaks by cellular processes such as replication. We used plasmid pBR322 and two‐dimensional agarose gel electrophoresis to examine the collision of replication forks with quinolone‐induced gyrase‐DNA cleavage complexes in Escherichia coli. Restriction endonuclease‐digested DNA exhibited a bubble arc with discrete spots, indicating that replication forks had been stalled. The most prominent spot depended upon the strong gyrase binding site of pBR322, providing direct evidence that quinolone‐induced cleavage complexes block bacterial replication forks in vivo. We differentiated between stalled forks that do or do not contain bound cleavage complex by extracting DNA under different conditions. Resealing conditions allow gyrase to efficiently reseal the transient breaks within cleavage complexes, while cleavage conditions cause the latent breaks to be revealed. These experiments showed that some stalled forks did not contain a cleavage complex, implying that gyrase had dissociated in vivo and yet the fork had not restarted at the time of DNA isolation. Additionally, some branched plasmid DNA isolated under resealing conditions nonetheless contained broken DNA ends. We discuss a model for the creation of double‐stranded breaks by an indirect mechanism after quinolone treatment.

Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase☆

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.

Replication, Recombination, and Repair: Going for the Gold

DNA recombination is now appreciated to be integral to DNA replication and cell survival. Recombination allows replication to successfully maneuver through the roadblocks of damaged or collapsed replication forks. The signals and controls that permit cells to transition between replication and recombination modes are now being identified.