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

SeqA protein negatively regulates replication initiation in Escherichia coli and is also proposed to organize maturation and segregation of the newly replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two‐ended breaks are expected. Instead, we show that, in SeqA's absence, chromosomes mostly suffer one‐ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants. Quantitative kinetics of origin and terminus replication from aligned chromosomes not only confirm origin overinitiation in seqA mutants, but also reveal terminus under‐replication, indicating inhibition of replication forks. Pre‐/post‐labelling studies of the chromosomal fragmentation in seqA mutants suggest events involving single forks, rather than pairs of forks from consecutive rounds rear‐ending into each other. We suggest that, in the absence of SeqA, the sister‐chromatid cohesion ‘safety spacer’ is destabilized and completely disappears if the replication fork is inhibited, leading to the segregation fork running into the inhibited replication fork and snapping the latter at single‐stranded DNA regions.     

A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation

SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery.     

The Escherichia coli SeqA protein

The Escherichia coli SeqA protein contributes to regulation of chromosome replication by preventing re-initiation at newly replicated origins. SeqA protein binds to new DNA which is hemimethylated at the adenine of GATC sequences. Most of the cellular SeqA is found complexed with the new DNA at the replication forks. In vitro the SeqA protein binds as a dimer to two GATC sites and is capable of forming a helical fiber of dimers through interactions of the N-terminal domain. SeqA can also bind, with less affinity, to fully methylated origins and affect timing of “primary” initiations. In addition to its roles in replication, the SeqA protein may also act in chromosome organization and gene regulation.     

Structural insights into the cooperative binding of SeqA to a tandem GATC repeat

SeqA is a negative regulator of DNA replication in Escherichia coli and related bacteria that functions by sequestering the origin of replication and facilitating its resetting after every initiation event. Inactivation of the seqA gene leads to unsynchronized rounds of replication, abnormal localization of nucleoids and increased negative superhelicity. Excess SeqA also disrupts replication synchrony and affects cell division. SeqA exerts its functions by binding clusters of transiently hemimethylated GATC sequences generated during replication. However, the molecular mechanisms that trigger formation and disassembly of such complex are unclear. We present here the crystal structure of a dimeric mutant of SeqA [SeqAΔ(41–59)-A25R] bound to tandem hemimethylated GATC sites. The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings. The SeqA–DNA complex also unveils additional protein–protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA. Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.     

Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork. We show that this is, indeed, the case by using global ChIP on Chip analysis of SeqA in cells synchronized regarding DNA replication. To assess hemimethylation, we developed the first genome-wide method for methylation analysis in bacteria. Since loss of the SeqA protein affects growth rate only during rapid growth when cells contain multiple replication forks, a comparison of rapid and slow growth was performed. In cells with six replication forks per chromosome, the two old forks were found to bind surprisingly little SeqA protein. Cell cycle analysis showed that loss of SeqA from the old forks did not occur at initiation of the new forks, but instead occurs at a time point coinciding with the end of SeqA-dependent origin sequestration. The finding suggests simultaneous origin de-sequestration and loss of SeqA from old replication forks.     

Characteristics of replication-independent endogenous double-strand breaks in Saccharomyces cerevisiae

Background

Replication-independent endogenous double-strand breaks (RIND-EDSBs) occur in both humans and yeast in the absence of inductive agents and DNA replication. In human cells, RIND-EDSBs are hypermethylated, preferentially retained in the heterochromatin and unbound by γ-H2AX. In single gene deletion yeast strains, the RIND-EDSB levels are altered; the number of RIND-EDSBs is higher in strains with deletions of histone deacetylase, endonucleases, topoisomerase, or DNA repair regulators, but lower in strains with deletions of the high-mobility group box proteins or Sir2. In summary, RIND-EDSBs are different from pathologic DSBs in terms of their causes and consequences. In this study, we identified the nucleotide sequences surrounding RIND-EDSBs and investigated the features of these sequences as well as their break locations.

Results

In recent work, we detected RIND-EDSBs using ligation mediated PCR. In this study, we sequenced RIND-EDSB PCR products of resting state Saccharomyces cerevisiae using next-generation sequencing to analyze RIND-EDSB sequences. We found that the break locations are scattered across a number of chromosomes. The number of breaks correlated with the size of the chromosomes. Most importantly, the break occurrences had sequence pattern specificity. Specifically, the majority of the breaks occurred immediately after the sequence “ACGT” (P = 2.2E-156). Because the “ACGT” sequence does not occur primarily in the yeast genome, this specificity of the “ACGT” sequence cannot be attributed to chance.

Conclusions

RIND-EDSBs occur non-randomly; that is, they are produced and retained by specific mechanisms. Because these particular mechanisms regulate their generation and they possess potentially specific functions, RIND-EDSBs could be epigenetic marks.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-750) contains supplementary material, which is available to authorized users.     

Ataxia Telangiectasia Mutated and Rad3 Related (ATR) Protein Kinase Inhibition Is Synthetically Lethal in XRCC1 Deficient Ovarian Cancer Cells

Introduction

Ataxia telangiectasia mutated and Rad3 Related (ATR) protein kinase is a key sensor of single-stranded DNA associated with stalled replication forks and repair intermediates generated during DNA repair. XRCC1 is a critical enzyme in single strand break repair and base excision repair. XRCC1-LIG3 complex is also an important contributor to the ligation step of the nucleotide excision repair response.

Methods

In the current study, we investigated synthetic lethality in XRCC1 deficient and XRCC1 proficient Chinese Hamster ovary (CHO) and human ovarian cancer cells using ATR inhibitors (NU6027). In addition, we also investigated the ability of ATR inhibitors to potentiate cisplatin cytotoxicity in XRCC1 deficient and XRCC1 proficient CHO and human cancer cells. Clonogenic assays, alkaline COMET assays, γH2AX immunocytochemistry, FACS for cell cycle as well as FITC-annexin V flow cytometric analysis were performed.

Results

ATR inhibition is synthetically lethal in XRCC1 deficient cells as evidenced by increased cytotoxicity, accumulation of double strand DNA breaks, G2/M cell cycle arrest and increased apoptosis. Compared to cisplatin alone, combination of cisplatin and ATR inhibitor results in enhanced cytotoxicity in XRCC1 deficient cells compared to XRCC1 proficient cells.

Conclusions

Our data provides evidence that ATR inhibition is suitable for synthetic lethality application and cisplatin chemopotentiation in XRCC1 deficient ovarian cancer cells.     

The effect of DNA-dependent protein kinase on adeno-associated virus replication

Background

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive.

Methodology/Principal Findings

In the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, while anti-Ku70 antibody moderately decreased rAAV replication. Similarly, when Ku heterodimer (Ku70/80) was depleted, less replicated rAAV DNA were detected. Finally, we showed that AAV-ITRs directly interacted with Ku proteins.

Conclusion/Significance

Collectively, our results showed that that DNA-PK enhances rAAV replication through the interaction of Ku proteins and AAV-ITRs.     

Lack of SeqA focus formation,specific DNA binding and proper protein multimerization in the Escherichia coli sequestration mutant seqA2

In Escherichia coli wild-type cells newly formed origins cannot be reinitiated. The prevention of reinitiation is termed sequestration and is dependent on the hemimethylated state of newly replicated DNA. Several mutants discovered in a screen for the inability to sequester hemimethylated origins have been mapped to the seqA gene. Here, one of these mutants, seqA2, harbouring a single amino acid change in the C-terminal end of the SeqA protein, was found to also be unable to form foci in vivo. The SeqA foci seen in the wild-type cells are believed to arise from multimerization of SeqA on hemimethylated DNA at the replication fork, presumably representing organization of newly formed DNA by SeqA. The result suggests that the process of origin sequestration is closely tied to the process of focus maintenance at the replication fork. In vitro, purified SeqA2 protein was found incapable of forming highly ordered multimers that bind hemimethylated oriC. The mutant protein was also incapable of restraining negative supercoils. Both in vivo and in vitro results support the idea that origin sequestration is an integral part of organization of newly formed DNA performed by SeqA.     

Impact of Acetazolamide and CPAP on Cortical Activity in Obstructive Sleep Apnea Patients

Study Objectives

1) To investigate the impact of acetazolamide, a drug commonly prescribed for altitude sickness, on cortical oscillations in patients with obstructive sleep apnea syndrome (OSAS). 2) To examine alterations in the sleep EEG after short-term discontinuation of continuous positive airway pressure (CPAP) therapy.

Design

Data from two double-blind, placebo-controlled randomized cross-over design studies were analyzed.

Setting

Polysomnographic recordings in sleep laboratory at 490 m and at moderate altitudes in the Swiss Alps: 1630 or 1860 m and 2590 m.

Patients

Study 1: 39 OSAS patients. Study 2: 41 OSAS patients.

Interventions

Study 1: OSAS patients withdrawn from treatment with CPAP. Study 2: OSAS patients treated with autoCPAP. Treatment with acetazolamide (500–750 mg) or placebo at moderate altitudes.

Measurements and Results

An evening dose of 500 mg acetazolamide reduced slow-wave activity (SWA; approximately 10%) and increased spindle activity (approximately 10%) during non-REM sleep. In addition, alpha activity during wake after lights out was increased. An evening dose of 250 mg did not affect these cortical oscillations. Discontinuation of CPAP therapy revealed a reduction in SWA (5–10%) and increase in beta activity (approximately 25%).

Conclusions

The higher evening dose of 500 mg acetazolamide showed the “spectral fingerprint” of Benzodiazepines, while 250 mg acetazolamide had no impact on cortical oscillations. However, both doses had beneficial effects on oxygen saturation and sleep quality.     

Regulation of Sister Chromosome Cohesion by the Replication Fork Tracking Protein SeqA

Analogously to chromosome cohesion in eukaryotes, newly replicated DNA in E. coli is held together by inter-sister linkages before partitioning into daughter nucleoids. In both cases, initial joining is apparently mediated by DNA catenation, in which replication-induced positive supercoils diffuse behind the fork, causing newly replicated duplexes to twist around each other. Type-II topoisomerase-catalyzed sister separation is delayed by the well-characterized cohesin complex in eukaryotes, but cohesion control in E. coli is not currently understood. We report that the abundant fork tracking protein SeqA is a strong positive regulator of cohesion, and is responsible for markedly prolonged cohesion observed at “snap” loci. Epistasis analysis suggests that SeqA stabilizes cohesion by antagonizing Topo IV-mediated sister resolution, and possibly also by a direct bridging mechanism. We show that variable cohesion observed along the E. coli chromosome is caused by differential SeqA binding, with oriC and snap loci binding disproportionally more SeqA. We propose that SeqA binding results in loose inter-duplex junctions that are resistant to Topo IV cleavage. Lastly, reducing cohesion by genetic manipulation of Topo IV or SeqA resulted in dramatically slowed sister locus separation and poor nucleoid partitioning, indicating that cohesion has a prominent role in chromosome segregation.     

miR-485-5p Binding Site SNP rs8752 in HPGD Gene Is Associated with Breast Cancer Risk

Background

Single nucleotide polymorphisms (SNPs) that reside in microRNA target sites may play an important role in breast cancer development and progression. To reveal the association between microRNA target site SNPs and breast cancer risk, we performed a large case-control study in China.

Methods

We performed a two-stage case-control study including 2744 breast cancer cases and 3125 controls. In Stage I, we genotyped 192 SNPs within microRNA binding sites identified from the “Patrocles” database using custom Illumina GoldenGate VeraCode assays on the Illumina BeadXpress platform. In Stage II, genotyping was performed on SNPs potentially associated with breast cancer risk using the TaqMan platform in an independent replication set.

Results

In stage I, 15 SNPs were identified to be significantly associated with breast cancer risk (P<0.05). In stage II, one SNP rs8752 was replicated at P<0.05. This SNP is located in the 3’ untranslated region (UTR) of the 15-hydroxyprostaglandin dehydrogenase (HPGD) gene at 4q34-35, a miR-485-5p binding site. Compared with the GG genotype, the combined GA+AA genotypes has a significantly higher risk of breast cancer (OR = 1.18; 95% CI: 1.06-1.31, P = 0.002). Specifically, this SNP was associated with estrogen receptor (ER) positive breast cancer (P = 0.0007), but not with ER negative breast cancer (P = 0.23), though p for heterogeneity not significant.

Conclusion

Through a systematic case-control study of microRNA binding site SNPs, we identified a new breast cancer risk variant rs8752 in HPGD in Chinese women. Further studies are warranted to investigate the underling mechanism for this association.     

Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism

Replication from the origin of Escherichia coli has traditionally been visualized as two replisomes moving away from each other, each containing a leading and a lagging strand polymerase. Fluorescence microscopy studies of tagged polymerases or forks have, however, indicated that the polymerases may be confined to a single location (or a few locations in cells with overlapping replication cycles). Here, we have analysed the exact replication patterns of cells growing with four different growth and replication rates, and compared these with the distributions of SeqA foci. The SeqA foci represent replication forks because the SeqA protein binds to the newly formed hemimethylated DNA immediately following the forks. The results show that pairs of forks originating from the same origin stay coupled for most of the cell cycle and thus support the replication factory model. They also suggest that the factories consisting of four polymerases are, at the time immediately after initiation, organized into higher order structures consisting of eight or 12 polymerases. The organization into replication factories was lost when replication forks experienced a limitation in the supply of nucleotides or when the thymidylate synthetase gene was mutated. These results support the idea that the nucleotide synthesis apparatus co-localizes with the replisomes forming a 'hyperstructure' and further suggest that the integrity of the replication factories and hyperstructures is dependent on nucleotide metabolism.     

Characterization of Nucleotide Misincorporation Patterns in the Iceman's Mitochondrial DNA

Background

The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation (“miscoding lesions”) has been the object of extensive investigations.

Methodology/Principal Findings

To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp) mitochondrial (mt) DNA sequences obtained from the 5,350–5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Ötzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp) mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85%) of the observed nucleotide misincorporations in ancient sequences.

Conclusions/Significance

This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine→thymine/guanine→adenine) transitions and that type 1 transitions are essentially PCR artifacts.     

Relative Influence of Genetics and Shared Environment on Child Mental Health Symptoms Depends on Comorbidity

Background

Comorbidity among childhood mental health symptoms is common in clinical and community samples and should be accounted for when investigating etiology. We therefore aimed to uncover latent classes of mental health symptoms in middle childhood in a community sample, and to determine the latent genetic and environmental influences on those classes.

Methods

The sample comprised representative cohorts of twins. A questionnaire-based assessment of mental health symptoms was used in latent class analyses. Data on 3223 twins (1578 boys and 1645 girls) with a mean age of 7.5 years were analyzed. The sample was predominantly non-Hispanic Caucasian (92.1%).

Results

Latent class models delineated groups of children according to symptom profiles–not necessarily clinical groups but groups representing the general population, most with scores in the normative range. The best-fitting models suggested 9 classes for both girls and boys. Eight of the classes were very similar across sexes; these classes ranged from a “Low Symptom” class to a “Moderately Internalizing & Severely Externalizing” class. In addition, a “Moderately Anxious” class was identified for girls but not boys, and a “Severely Impulsive & Inattentive” class was identified for boys but not girls. Sex-combined analyses implicated moderate genetic influences for all classes. Shared environmental influences were moderate for the “Low Symptom” and “Moderately Internalizing & Severely Externalizing” classes, and small to zero for other classes.

Conclusions

We conclude that symptom classes are largely similar across sexes in middle childhood. Heritability was moderate for all classes, but shared environment played a greater role for classes in which no one type of symptom predominated.     

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

TrAEL-seq provides genome-wide base pair resolution maps of exposed DNA 3’ ends; this reveals replication fork stalling and normal replication profiles in asynchronous, unlabelled wildtype cell populations, along with the sites of resected DNA breaks.