Replication fork inhibition in <scp>seqA</scp> mutants of <scp>E</scp>scherichia coli triggers replication fork breakage

Rotman, Ella; Khan, Sharik R.; Kouzminova, Elena A.; Kuzminov, Andrei

doi:10.1111/mmi.12638

Cited by 19 publications

(39 citation statements)

References 86 publications

(178 reference statements)

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“…We concluded that, while the rapidly growing wild-type cells have a single replication round per chromosome ( Figure 1E, the blue profile), cells with inhibited replication forks maintain up to three replication rounds in their chromosome (Figure 1, E and F). Since three replication rounds per chromosome is the highest replication complexity in growing cells reported in the literature (Lane and Denhardt 1975;Martín and Guzmán 2011;Rotman et al 2014), both our and the literature data suggest "CRC8" in E. coli as a natural limit showing static features. …”

mentioning

confidence: 81%

“…Indeed, both the HU treatment, on the one hand, and the seqA and rep defects, on the other hand, induce formation of double-strand DNA breaks (Michel et al 1997;Mohana-Borges et al 2000;Rotman et al 2014), perhaps the most serious of all chromosome lesions (Resnick 1978;Iliakis 1991). At the same time, both the natural CRC8 and the functional CRC22 limits are well-tolerated, suggesting robust chromosomal repair mechanisms.…”

Section: A System To Maximize Crcmentioning

confidence: 93%

“…Inability of replication forks to reach the terminus means that they are inactivated. Since the obvious type of chromosomal condition due to high CRC is "replication fork crowding" (Table S2), the suspected major consequence would be replication forks rear-ending into each other to generate double-strand DNA breaks (Bidnenko et al 2002;Rotman et al 2014) (Figure 4B). Thus, the inability of replication forks to reach the terminus under conditions of runaway CRC may be similar to the situation in the dut recBC mutants, where replication forks succumb to chromosome fragmentation detectable in pulsed-field gels (Kouzminova and Kuzminov 2006).…”

Section: Runaway Crc Fragments the Chromosomementioning

confidence: 99%

“…HU was then added to 10 mM, and the cultures were grown for an additional 4 hr at 28°. Samples were taken at time 0 (before HU addition) and after 4 hr, and the ori/ter ratio in the phenol-chloroform-purified chromosomal DNA from these cultures was measured using Southern hybridization, as before (Kouzminova and Kuzminov 2006;Rotman et al 2014). Overnight (ori/ter = 1) and exponentially growing (ori/ter = 2) AB1157 cultures offered controls.…”

Section: Viability Assaysmentioning

confidence: 99%

“…Perhaps thymine starvation is an exception to its sensitivity to increased CRC, while the eclipse phenomenon can be thought of as merely a time measure that standardizes distance between consecutive replication forks when fork progression is slowed, as in seqA mutants (Olsson et al 2002). On the other hand, E. coli mutants with increased CRC suffer from increased chromosomal fragmentation (Michel et al 1997;Rotman et al 2014), while in humans increased CRC is linked to chromosome rearrangements (Hastings et al 2009;Zhang et al 2009) and carcinogenesis (Hook et al 2007;Truong et al 2014), suggesting that increased CRC is an important factor of genome instability.The initial objectives of this study were, by pushing E. coli cells to maximize chromosomal replication complexity (in the rep and seqA mutants or in HU-treated cells): (1) to confirm the maximal chromosomal replication complexity that E. coli can routinely achieve without major adverse consequences-the natural CRC limit; (2) to determine the maximal chromosomal replication complexity at which E. coli can still slowly grow-the functional CRC limit; (3) to push E. coli to develop the maximal chromosomal replication complexity that the cells can survive-the tolerance CRC limit; (4) to understand the nature of growth inhibition due to the increased CRC. In our efforts to maximize CRC, we specifically avoided the subject of regulation of the replication initiation at the chromosomal origin and, in fact, removed this consideration altogether in subsequent experimentation by initiating chromosome replication from an inducible plasmid replication origin.…”

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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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mentioning

confidence: 81%

Section: A System To Maximize Crcmentioning

confidence: 93%

Section: Runaway Crc Fragments the Chromosomementioning

confidence: 99%

Section: Viability Assaysmentioning

confidence: 99%

mentioning

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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Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

2015

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In E. coli, errors in newly-replicated DNA, such as the incorporation of a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutHas they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemimethylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.

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Prompt repair of hydrogen peroxide-induced DNA lesions prevents catastrophic chromosomal fragmentation

Mahaseth

Kuzminov

2016

DNA Repair

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Iron-dependent oxidative DNA damage in vivo by hydrogen peroxide (H2O2, HP) induces copious single-strand(ss)-breaks and base modifications. HP also causes infrequent double-strand DNA breaks, whose relationship to the cell killing is unclear. Since hydrogen peroxide only fragments chromosomes in growing cells, these double-strand breaks were thought to represent replication forks collapsed at direct or excision ss-breaks and to be fully reparable. We have recently reported that hydrogen peroxide kills Escherichia coli by inducing catastrophic chromosome fragmentation, while cyanide (CN) potentiates both the killing and fragmentation. Remarkably, the extreme density of CN+HP-induced chromosomal double-strand breaks makes involvement of replication forks unlikely. Here we show that this massive fragmentation is further amplified by inactivation of ss-break repair or base-excision repair, suggesting that unrepaired primary DNA lesions are directly converted into double-strand breaks. Indeed, blocking DNA replication lowers CN+HP-induced fragmentation only ~2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend ~100 breaks spread over the entire chromosome. Therefore, double-strand breaks induced by oxidative damage happen at the sites of unrepaired primary one-strand DNA lesions, are independent of replication and are highly lethal, supporting the model of clustered ss-breaks at the sites of stable DNA-iron complexes.

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Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage

Cited by 19 publications

References 86 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

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

Prompt repair of hydrogen peroxide-induced DNA lesions prevents catastrophic chromosomal fragmentation

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