RecA-dependent mutants in
            <i>Escherichia coli</i>
            reveal strategies to avoid chromosomal fragmentation

Kouzminova, Elena A.; Rotman, Ella; Macomber, Lee; Zhang, Jian; Kuzminov, Andrei

doi:10.1073/pnas.0405943101

Cited by 61 publications

(101 citation statements)

References 50 publications

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“…Rad51, Rad52, and the Mre11 complex all are involved in recombinational DSB repair (40), consistent with the view that recombination is required for repairing the lesions that occur in tas1 mutants. Interestingly, in Escherichia coli, mutation fur, ubiH, or ubiE, most likely leading to an increased level of ROS, causes cell death in the absence of RecA-dependent recombinational repair (41). The fur recA synthetic lethality is ascribed to elevated chromosomal fragmentation.…”

Section: Discussionmentioning

confidence: 99%

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

Ragu

Faye

Iraqui

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The absence of Tsa1, a key peroxiredoxin that functions to scavenge H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the activity of the translesion DNA polymerases , , and Rev1. Anaerobic growth reduced substantially GCR rates of WT and tsa1 mutants and restored the viability of tsa1 rad6, tsa1 rad51, and tsa1 mre11 double mutants. Anaerobic growth also reduced the GCR rate of rad27, pif1, and rad52 mutants, indicating a role of reactive oxygen species in GCR formation in these mutants. In addition, deletion of TSA1 or H 2O2 treatment of WT cells resulted in increased formation of Rad52 foci, sites of repair of multiple DNA lesions. H 2O2 treatment also induced the GCRs. Our results provide in vivo evidence that oxygen metabolism and reactive oxygen species are important sources of DNA damages that can lead to GCRs and lethal effects in S. cerevisiae.dsDNA break ͉ gross chromosomal rearrangement ͉ translesion DNA synthesis ͉ peroxiredoxin M aintaining genome stability is crucial for cell growth and cell survival. Different genetic disorders, including most human cancers, are associated with different forms of genome instability (1-3). One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangements (GCRs), such as translocations, deletions of chromosome arms, interstitial deletions, inversions, amplifications, chromosome fusions, and aneuploidy. Multiple genes and pathways that suppress GCRs have been characterized by using the yeast Saccharomyces cerevisiae as a model system (4-6). These include genes involved in DNA replication, recombination, and repair, cell-cycle checkpoints that function during DNA replication and repair, and pathways involved in telomere maintenance, chromatin assembly, and detoxification of reactive oxygen species (ROS) (refs. 7 and 8 and references therein). The importance of these pathways for suppressing genome instability is further emphasized by their roles in preventing the development of human cancer (7, 9, 10).Although many pathways that function in the suppression of GCRs have been discovered, little is known about the causes and origin of GCRs. It is generally thought that a major cause of DNA damage that leads to mutations is ROS, which are generated as a normal part of oxygen metabolism but are also produced by ionizing radiation, metabolism of exogenous compounds, and pathological processes such as infection and inflammation (11,12). ROS, such as the superoxide radical, H 2 O 2 , and the hydroxyl radical, can attack almost all cell components and can induce many types of DNA damage, including single-stranded DNA breaks and dsDNA breaks (DSB), base and sugar modificat...

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Section: Discussionmentioning

confidence: 99%

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

Ragu

Faye

Iraqui

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…CZ, compression zone. The resolution of these gels is such that the bottom corresponds to 50 kbp, while the compression zone accumulates species that are $2 mbp (Kouzminova et al 2004). (D) Quantification of chromosomal fragmentation at 3 hr (3 h) and 6 hr (6 h) of IPTG induction in various mutants carrying inducible origin.…”

Section: Runaway Crc Fragments the Chromosomementioning

confidence: 99%

“…Because we suspected that high CRC induces chromosome fragmentation, making cells dependent on double-strand break repair catalyzed in E. coli by the RecBCD pathway (Kuzminov 1999, 2011), we could not genetically explore high CRC tolerance using the seqA and rep mutants, as both depend on the RecBCD pathway for viability (Uzest et al 1995; Kouzminova et al 2004). Therefore, to specifically address our new objectives, we built an experimental system with conditional (inducible) high CRC by inserting an inducible bidirectional replication origin near oriC ( Figure 3A).…”

Section: A System To Maximize Crcmentioning

confidence: 99%

“…To detect low-level chromosomal fragmentation in E. coli, both linear DNA degradation and its recombinational repair have to be blocked with a recBC mutation (Michel et al 1997;Kouzminova et al 2004;Khan and Kuzminov 2012) ( Figure 5A). Indeed, we found modest levels of chromosomal fragmentation [reaching 15% over the background after 5-6 hr of IPTG induction ( Figure S5)] to develop in the IOC recBC mutant, compared to ,5% in the wild-type cells, recA, ruvABC, and seqA mutants (Figure 4, C and D).…”

Section: Runaway Crc Fragments the Chromosomementioning

confidence: 99%

“…Our three new objectives were to investigate the following: (1) the nature of chromosomal lesions associated with high CRC; (2) the mechanisms (avoidance or repair) allowing cells to tolerate high CRC; (3) the nature of irreparable chromosomal damage caused by the maximal CRC. The third objective reflected our expectation that breaking through the functional CRC22 limit, if proved possible, would result in cell lethality due to particular chromosomal problems, which we could characterize.Because we suspected that high CRC induces chromosome fragmentation, making cells dependent on double-strand break repair catalyzed in E. coli by the RecBCD pathway (Kuzminov 1999, 2011), we could not genetically explore high CRC tolerance using the seqA and rep mutants, as both depend on the RecBCD pathway for viability (Uzest et al 1995; Kouzminova et al 2004). Therefore, to specifically address our new objectives, we built an experimental system with conditional (inducible) high CRC by inserting an inducible bidirectional replication origin near oriC ( Figure 3A).…”

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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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Diverse roles of nucleoside diphosphate kinase in genome stability and growth fitness

Kapoor

Varshney

2020

Curr Genet

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RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation

Cited by 61 publications

References 50 publications

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

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

Diverse roles of nucleoside diphosphate kinase in genome stability and growth fitness

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