The role of replication initiation control in promoting survival of replication fork damage

Sutera, Vincent A.; Lovett, Susan T.

doi:10.1111/j.1365-2958.2006.05093.x

Cited by 43 publications

(44 citation statements)

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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%

“…Below our experiments with mutants, conditions and artificial constructs are matched with the contrasting expectations of the static vs. dynamic models of CRC regulation in E. coli. The slow-growing seqA mutants are sensitive to HU (Sutera and Lovett 2006) and are synergistically inhibited by 1 mM HU, the concentration at which wild-type cell growth is unaffected (Figure 2A). To test the static nature of CRC8 limit, we inhibited DNA synthesis in seqA and rep single mutants, as well as in seqA rep double mutants, with 10 mM HU for 4 hr.…”

Section: Predictions Of Static Vs Dynamic Crc Regulation Modelsmentioning

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: Predictions Of Static Vs Dynamic Crc Regulation Modelsmentioning

confidence: 99%

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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“…All plasmids in this study were constructed using Gateway Cloning Technology (Invitrogen) from PCR products amplified using MG1655 chromosomal template DNA (Masterpure DNA purification kit; Epicentre) and Pfu Turbo (Stratagene) or Phusion polymerase (Finnzymes) and were recovered by transformation into E. coli K-12 strain DH5␣. After purification (PCR purification kit; Qiagen), sequences were inserted into Gateway high-copy vector pDONR201, conferring kanamycin resistance, by the BP reaction (Invitrogen) and by LR reaction into destination vectors pSTL358 (38) and pDEW201::GW (34), respectively, to generate low-copy complementation plasmids and lux reporters. Plasmid pDONR-P-iraD carrying iraD ORF and its upstream region were constructed using the primers 5Ј GGGGACAAGT TTG-TACAAAA AAGCAGGCTT CGAAGGAGAT AGAACCGTAA ACAAATGACA TG-CATGTTTCT and 5Ј GGGGACCACT TTGTACAAGA AAGCTGGGTC TTAGCT-GACA TTCTCCAGCG TCGCACTGCG.…”

Section: Methodsmentioning

confidence: 99%

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS

Merrikh

Ferrazzoli

Bougdour

et al. 2009

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

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We isolated an Escherichia coli mutant in the iraD gene, sensitive to various forms of DNA damage. Our data are consistent with the function of IraD to promote accumulation of the alternative transcription sigma factor, RpoS, by binding to the adaptor RssB protein that targets RpoS for degradation. Our results demonstrate the physiological importance of this mode of regulation for DNA damage tolerance. Although RpoS is best known for its regulation of genes induced in stationary phase, our work underscores the importance of the RpoS regulon in a DNA damage response in actively growing cells. We show that iraD transcription is induced by DNA damage by a mechanism independent of the SOS response. The IraD and SOS regulatory pathways appear to act synergistically to ensure survival of cells faced with oxidative or DNA damaging stress during cellular growth.oxidative stress ͉ posttranslational regulation ͉ replication stress ͉ SOS response ͉ DNA repair T hroughout its life cycle, Escherichia coli is faced with different environmental challenges and regulates gene expression accordingly. One way is by changes in the promoter recognition of RNA polymerase via different situation-specific factors (1). In E. coli, the major alternative sigma factor is S (RpoS), which is required for expression of specific genes on entry to stationary phase or as a response to stress (2-4). Although the RpoS dependence of many of these responses and the regulation of RpoS itself have been well studied, the relevance of this to DNA repair has not been a major focus.To find genes important in DNA damage responses, we performed a random Tn5 transposon insertion mutant screen, assaying sensitivity to, among other agents, phleomycin and azidothymidine (AZT). Phleomycin induces random single-or double-strand breaks in the backbone of DNA (5), whereas AZT blocks DNA synthesis, leading to single-strand gaps in the replication fork (6). One insertion mutant in iraD (previously an unknown gene, yjiD) was hypersensitive to phleomycin and AZT.Recent work from Gottesman and coworkers (7) implicated IraD in posttranslational regulation of RpoS. The RssB adaptor protein targets RpoS to ClpXP for degradation during logarithmic growth, keeping RpoS protein levels low in the absence of stress (8-12). IraD was identified in a high-copy plasmid screen for genes promoting accumulation of an RpoS-LacZ fusion protein. The IraD gene product acts as an antiadaptor protein via direct binding and inhibition of the ability of RssB to target RpoS for proteolysis by ClpXP in vitro (7).In the work presented here, we demonstrate that IraD is required for survival to DNA damage, providing evidence of the physiological importance of IraD in particular and the antiadaptor mechanism in general. The data presented suggest that IraD acts as an antagonist of RssB, regulating RpoS levels and stabilization, not only after DNA damage but constitutively. Our results establish the importance of RpoS stabilization in proliferating bacterial cells in which replication has been direc...

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“…A mutation in dnaA is necessary and sufficient for suppression of seqA: Because dnaA and seqA mutations are mutually suppressive (Lu et al 1994;von Freiesleben et al 1994;Sutera and Lovett 2006), we performed genetic crosses to establish whether two independently isolated oss mutations mapped near dnaA. We crossed seqA obgE oss strains with a selectable marker, zid-501T Tn10, 86% cotransducible with dnaA.…”

Section: Resultsmentioning

confidence: 99%

“…We became interested in SeqA while studying factors that promoted survival to chronic exposure to low levels of replication inhibitors (Sutera and Lovett 2006). Mutants in seqA and dam were sensitive to such agents, such as hydroxyurea and azidothymidine; this sensitivity was exacerbated under fast-growth conditions during which E. coli has multiple ongoing replication cycles.…”

Section: R Eplication Initiation In Bacteria Is Controlled Bymentioning

confidence: 99%

A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

Molt

Sutera

Moore³

et al. 2009

Genetics

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The initiation of replication in bacteria is regulated via the initiator protein DnaA. ATP-bound DnaA binds to multiple sequences at the origin of replication, oriC, unwinding the DNA and promoting the binding of DnaB helicase. From an Escherichia coli mutant highly perturbed for replication control, obgETTn5-EZ seqAD, we isolated multiple spontaneous suppressor mutants with enhanced growth and viability. These suppressors suppressed the replication control defects of mutants in seqA alone and genetically mapped to the essential dnaA replication initiator gene. DNA sequence analysis of four independent isolates revealed an identical deletion of the DnaA-coding region at a repeated hexanucleotide sequence, causing a loss of 25 amino acids in domain II of the DnaA protein. Previous work has established no function for this region of protein, and deletions in the region, unlike other domains of the DnaA protein, do not produce lethality. Flow cytometric analysis established that this allele, dnaAD 96-120 , ameliorated the over-replication phenotype of seqA mutants and reduced the DNA content of wild-type strains; virtually identical effects were produced by loss of the DnaA-positive regulatory protein DiaA. DiaA binds to multiple DnaA subunits and is thought to promote cooperative DnaA binding to weak affinity DNA sites through interactions with DnaA in domains I and/or II. The dnaAD 96-120 mutation did not affect DiaA binding in pull-down assays, and we propose that domain II, like DiaA, is required to promote optimal DnaB recruitment to oriC.

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The role of replication initiation control in promoting survival of replication fork damage

Cited by 43 publications

References 44 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

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS

A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

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