Chromosome demise in the wake of ligase‐deficient replication

Kouzminova, Elena A.; Kuzminov, Andrei

doi:10.1111/j.1365-2958.2012.08076.x

Cited by 27 publications

(50 citation statements)

References 77 publications

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“…Stalling of replication forks or accumulation of single-strand interruptions behind the forks in bacteria, therefore, leads to catastrophic chromosomal consequences, reflected in such phenomena as thymineless death (Kuong and Kuzminov 2012), ligase-deficient death (Kouzminova and Kuzminov 2012), or sensitivity of seqA mutants to DNA damage (Sutera and Lovett 2006) and to rapid growth . If chromosome segregation is still concurrent with replication in the cells with elevated CRC, the viability is expected to be unaffected, as is indeed observed at the natural and functional CRC limits.…”

Section: Discussionmentioning

confidence: 99%

“…Plug hybridization for ori and ter signals Plug hybridization followed the published protocol (Kouzminova and Kuzminov 2012). The plugs with unlabeled chromosomal DNA were washed in glass tubes with two changes of 1 ml TE (30 min each) on a rotary shaker with gentle agitation.…”

Section: Chromosomal Fragmentation Analysismentioning

confidence: 99%

“…After the final wash, DNA from plugs was vacuum-transferred in two identical sets onto nylon membrane (Hybond H+, GE Biosciences) and UV-crosslinked to the membrane (UV Crosslinker FB-UVXL-1000, Fisher). After UV treatment, the membrane was cut into two halves to separate the sets of transferred DNA, and one-half was probed with a 32 P-labeled oriC probe, while the other half with the 32 P-labeled dif probe (Kouzminova and Kuzminov 2012). Hybridization was performed overnight at 63°in a 0.5 M sodium phosphate and 5% SDS hybridization buffer.…”

Section: Chromosomal Fragmentation Analysismentioning

confidence: 99%

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Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Khan¹,

Mahaseth²,

Kouzminova³

et al. 2016

Genetics

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We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of 8 (cells divide more slowly); the "functional" CRC limit of 22 (cells divide extremely slowly); and the "tolerance" CRC limit of 64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter. KEYWORDS overinitiation; hydroxyurea; seqA; rep; recA E UKARYOTIC and prokaryotic chromosomes differ in many important aspects (Kuzminov 2014), and one key difference lies in the spatio-temporal organization of chromosomal replication. In contrast to eukaryotes, which perform multibubble replication (Masai et al. 2010), most bacteria replicate their singular chromosome in the unibubble format by initiating bidirectional replication from a designated replication origin (oriC) (Sernova and Gelfand 2008;Leonard and Méchali 2013) and finishing replication within a broad termination zone (ter) (Mirkin and Mirkin 2007;Duggin et al. 2008). Eukaryotes always perform a single replication round in their chromosomes (Masai et al. 2010;Diffley 2011), keeping the ratio of maximally replicated to unreplicated DNA in the same chromosome (the "replication complexity index") strictly at 2 ( Figure 1A). Due to the defined origin and terminus of prokaryotic chromosomes, chromosomal replication complexity in bacteria can be simply expressed as the ori/ter ratio ( Figure 1B). Even though unique cell cycles in some bacteria, such as Caulobacter, also maintain a strict CRC = 2 (Collier 2012), bacterial cells are generally recognized for their ability to support multiple concurrent replication rounds within the same chromosome (Morigen et al. 2009).In reality, under typical growth conditions the ori/ter ratio in exponentially growing bacterial cells is still 2 (Bird et al. 1972;Bipatnath et al. 1998;Wang et al. 2007;Murray and Errington 2008;Rotman et al. 2009;Stokke et al. 2011), showing that bacterial cells, like eukaryotes, also prefer to deal with a single replication round in their chromosomes. However, due to the peculiarity of the prokaryotic chromosome cycle (Kuzminov 2013), whe...

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Section: Discussionmentioning

confidence: 99%

Section: Chromosomal Fragmentation Analysismentioning

confidence: 99%

Section: Chromosomal Fragmentation Analysismentioning

confidence: 99%

See 1 more Smart Citation

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Khan¹,

Mahaseth²,

Kouzminova³

et al. 2016

Genetics

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“…The in vitro rate of purified main bacterial replicase DNA polymerase (pol) III is ϳ500 nt/s (155)(156)(157). The directly measured rates of replication fork propagation in vivo, at 620 to 700 nt/s at 30°C (158, 159) and 1,300 nt/s at 42°C (159), are even higher, and there is evidence that the rate is limited by the rate of DNA pol III chain elongation (160). In contrast, the maximal rate of the yeast leading-strand DNA polymerase epsilon in vitro is only 50 nt/s, although under the same conditions the lagging-strand DNA polymerase delta moves faster, at 200 nt/s (157).…”

Section: Prokaryotic Chromosome Organization Compensates For the Singmentioning

confidence: 99%

The Precarious Prokaryotic Chromosome

Kuzminov

2014

J Bacteriol

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Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the two distinct ways to organize chromosomes are driven by the differences between the global-consecutive chromosome cycle of eukaryotes and the local-concurrent chromosome cycle of prokaryotes. Specifically, progressive chromosome segregation in prokaryotes demands a single duplicon per chromosome, while other "precarious" features of the prokaryotic chromosomes can be viewed as compensations for this severe restriction.

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“…This enzyme is responsible for catalyzing the formation of a phosphodiester bond between the 5= phosphate of one nucleotide and the 3= hydroxyl group of another in the backbone of a DNA strand. The loss of DNA ligase function causes quick formation of double-strand breaks in the DNA structure, followed by exonucleolytic degradation of the fragmented DNA (20). For this reason, DNA ligase has a crucial role in DNA replication, recombination, and repair.…”

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Temperature Sensitivity Conferred by ligA Alleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

Pankowski

Puckett

Nano

2016

Appl Environ Microbiol

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We have assembled a collection of 13 psychrophilic ligA alleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (TS) phenotype. When these ligA alleles were substituted into Francisella novicida, they conferred a TS phenotype with restrictive temperatures between 33 and 39°C. When the F. novicida ligA hybrid strains were plated above their restrictive temperatures, eight of them generated temperature-resistant variants. For two alleles, the mutations that led to temperature resistance clustered near the 5= end of the gene, and the mutations increased the predicted strength of the ribosome binding site at least 3-fold. Four F. novicida ligA hybrid strains generated no temperature-resistant variants at a detectable level. These results suggest that multiple mutations are needed to create temperature-resistant variants of these ligA gene products. One ligA allele was isolated from a Colwellia species that has a maximal growth temperature of 12°C, and this allele supported growth of F. novicida only as a hybrid between the psychrophilic and the F. novicida ligA genes. However, the full psychrophilic gene alone supported the growth of Salmonella enterica, imparting a restrictive temperature of 27°C. We also tested two ligA alleles from two Pseudoalteromonas strains for their ability to support the viability of a Saccharomyces cerevisiae strain that lacked its essential gene, CDC9, encoding an ATP-dependent DNA ligase. In both cases, the psychrophilic bacterial alleles supported yeast viability and their expression generated TS phenotypes. This collection of ligA alleles should be useful in engineering bacteria, and possibly eukaryotic microbes, to predictable TS phenotypes.O ne aspect of synthetic biology is the development of genetic elements that can be used for genome engineering (1). These elements include promoters (2, 3), transcriptional enhancers (4), transcriptional stop elements (5), riboswitches (6), or site-specific recombinases (7). Some include reporter genes that encode fluorescent proteins, pigments, or odors (8). Others (9, 10) have proposed that essential genes could be a useful class of genetic elements that might be widely used to engineer the limits of viability of a variety of microbes under a specified restrictive condition, such as high temperature. One application of temperature restriction of growth could be in creating bacterial pathogens that are identical to the wild type in every trait except for growth above a defined temperature, such as 35°C. Such pathogens could be used in research, teaching, and diagnostic antigen preparation with minimal chance of causing invasive disease in humans. Another application is in creating attenuated vaccines, where temperature sensitivity is already a well-established approach for attenuation.Essential genes are defined as those that are required for the viability of an organism under all growth conditions (11-13). Biologists try to determine an organism's complement of essential genes for the inherent interest in...

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Chromosome demise in the wake of ligase‐deficient replication

Cited by 27 publications

References 77 publications

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

The Precarious Prokaryotic Chromosome

Temperature Sensitivity Conferred by ligA Alleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

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