Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics

Transcription Initiation Activity Sets Replication Origin Efficiency in Mammalian Cells

Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS), suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation, unveiling a new hallmark in ORI efficiency regulation in mammalian cells

Učinak bakra na toksičnost i genotoksičnost kadmija u vodenoj leći (Lemna minor L.)

We investigated interactions between copper (in the concentrations of 2.5 μmol L-1 and 5 μmol L-1) and cadmium (5 μmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 μmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 μmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 μmol L-1 copper but decreased in plants treated with cadmium and 5 μmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction.U svrhu istraživanja interakcija između bakra kao esencijalnog elementa te kadmija kao neesencijalnog i toksičnog metala, vodenu leću Lemna minor L. uzgajali smo na podlogama s kadmijem (5 μmol L-1) odnosno s bakrom (2,5 μmol L-1 i 5 μmol L-1) te s njihovim kombinacijama. Unos metala u biljke povećavao se s trajanjem pokusa, a kod kombinacije metala u biljkama je izmjerena niža količina kadmija nego u onima uzgajanima samo na kadmiju. U biljkama tretiranim pojedinačnim metalom došlo je do povećanja sadržaja malondialdehida (MDA) te aktivnosti katalaze i peroksidaze u odnosu na kontrolne biljke. Također, primijećeno je oštećenje DNA iako kod bakra tek sedmog dana tretmana. Količina MDA i aktivnost obaju enzima dodatno se povećala na tretmanu kombinacijom kadmija i bakra (5 μmol L-1) nakon četvrtog dana pokusa, dok se količina MDA smanjila nakon sedmog dana kod kombinacije kadmija i 2,5 μmol L-1 bakra. U tim biljkama primijećena je i veća aktivnost katalaze, dok je aktivnost peroksidaze porasla na tretmanu kadmijem i 2,5 μmol L-1 bakrom, ali se smanjila na tretmanu kadmijem i 5 μmol L-1 bakrom. Oštećenje DNA koje je bilo veće kod kombinacije metala nakon četvrtog dana, osobito u usporedbi sa samim bakrom, smanjilo se nakon sedmog dana pokusa. Iz ovih rezultata može se zaključiti da su oba metala u istraživanim koncentracijama toksična i genotoksična za vodenu leću i da uzrokuju oksidacijski stres. Kadmij u kombinaciji s bakrom više koncentracije bio je toksičniji od pojedinačnih metala nakon četvrtog dana pokusa, dok su u biljaka tretiranih kombinacijom kadmija i bakra niže koncentracije toksični učinci bili manji. Budući da su primijećene interakcije vrlo kompleksne i ne uključuju samo antagonizam odnosno sinergizam potrebna su daljnja istraživanja