Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase

The dnaQ gene of Escherichia coli encodes the Ɛ subunit of DNA polymerase III, which provides the 3\u27 - 5\u27 exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of Ɛ leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our laboratory identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response after quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby, in addition to proofreading, Ɛ plays a distinct role in replisome disassembly and/or processing of stalled replication forks. To explore this model, we generated a pentapeptide insertion mutant library of the dnaQgene, along with site-directed mutants, and screened for separation of function mutants. We report the identification of separation of function mutants from this screen, showing that proofreading function can be uncoupled from SOS phenotypes (partially constitutive SOS and the nalidixic acid SOS defect). Surprisingly, the two SOS phenotypes also appear to be separable from each other. These findings support the hypothesis that Ɛ has additional roles aside from proofreading. Identification of these mutants, especially those with normal proofreading but SOS phenotype(s), also facilitates the study of the role of e in SOS processes without the confounding results of high mutator activity associated with dnaQ knockout mutants

Initiation of bacteriophage T4 DNA replication and replication fork dynamics: a review in the Virology Journal series on bacteriophage T4 and its relatives

Abstract Bacteriophage T4 initiates DNA replication from specialized structures that form in its genome. Immediately after infection, RNA-DNA hybrids (R-loops) occur on (at least some) replication origins, with the annealed RNA serving as a primer for leading-strand synthesis in one direction. As the infection progresses, replication initiation becomes dependent on recombination proteins in a process called recombination-dependent replication (RDR). RDR occurs when the replication machinery is assembled onto D-loop recombination intermediates, and in this case, the invading 3' DNA end is used as a primer for leading strand synthesis. Over the last 15 years, these two modes of T4 DNA replication initiation have been studied in vivo using a variety of approaches, including replication of plasmids with segments of the T4 genome, analysis of replication intermediates by two-dimensional gel electrophoresis, and genomic approaches that measure DNA copy number as the infection progresses. In addition, biochemical approaches have reconstituted replication from origin R-loop structures and have clarified some detailed roles of both replication and recombination proteins in the process of RDR and related pathways. We will also discuss the parallels between T4 DNA replication modes and similar events in cellular and eukaryotic organelle DNA replication, and close with some current questions of interest concerning the mechanisms of replication, recombination and repair in phage T4.</p

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

Fork regression is an active helicase-driven pathway in bacteriophage T4

Reactivation of stalled replication forks requires specialized mechanisms that can recognize the fork structure and promote downstream processing events. Fork regression has been implicated in several models of fork reactivation as a crucial processing step that supports repair. However, it has also been suggested that regressed forks represent pathological structures rather than physiological intermediates of repair. To investigate the biological role of fork regression in bacteriophage T4, we tested several mechanistic models of regression: strand exchange-mediated extrusion, topology-driven fork reversal and helicase-mediated extrusion. Here, we report that UvsW, a T4 branch-specific helicase, is necessary for the accumulation of regressed forks in vivo, and that UvsW-catalysed regression is the dominant mechanism of origin-fork processing that contributes to double-strand end formation. We also show that UvsW resolves purified fork intermediates in vitro by fork regression. Regression is therefore part of an active, UvsW-driven pathway of fork processing in bacteriophage T4

Summary of growth in the presence of quinolones.

<p>For ease of comparison, the results of dilution plating (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128092#pone.0128092.g004" target="_blank">Fig 4</a>) were categorized in comparison to the wild-type dilution series on the same plate. The number in each entry indicates which dilution tube showed growth (more than one colony) relative to the wild-type series (+1 indicates that the mutant grew in the next more dilute spot than the wild type while -1 indicates that the mutant grew in the next more concentrated spot; note that each dilution step was five-fold, and so a reading of -3 would correspond to a dilution factor differential of 125). The downward arrow next to the number indicates that the colonies that did grow were noticeably smaller than the corresponding wild-type colonies on the same plate (or that no colonies grew in the corresponding mutant spot); the upward arrow for the <i>ΔrecF</i> spots indicated that the colonies grew larger than the corresponding wild-type colonies. Both negative numbers and downward arrows provide evidence for hypersensitivity.</p><p>* Mutants that we designated as hypersensitive are indicated by an asterisk.</p><p>^a Ciprofloxacin, cipro</p><p>^b Nalidixic acid, nal</p><p>Summary of growth in the presence of quinolones.</p