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Background : It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented. Methods : We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome. Results : The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the 'domino' model of replication focus activation order observed by microscopy. Conclusions : We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 da Costa-Nunes JA et al.)

Background: The RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported., Results: We show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role in somatic DNA dsb repair. Comet data shows that the lack of either cohesins induces a similar high basal level of DNA dsb in the nuclei and a slower DNA dsb repair kinetics in both cohesin mutants. The observed AtRAD21.3 transcriptional response to DNA dsb induction reinforces further the role of this cohesin in DNA dsb repair. The importance of AtRAD21.3 in DNA dsb damage repair, after exposure to DNA dsb damage inducing agents, is notorious and recognisably evident at the phenotypical level, particularly when the AtRAD21.1 gene is also disrupted., Conclusions: Our data demonstrates that both Arabidopsis cohesin (AtRAD21.1 and AtRAD21.3) play a role in somatic DNA dsb repair. Furthermore, the phenotypical data from the atrad21.1 atrad21.3 double mutant indicates that these two cohesins function synergistically in DNA dsb repair. The implications of this data are discussed.

The RAD21/REC8 gene family has been implicated in sister chromatid cohesion and DNA repair in several organisms. Unlike most eukaryotes, Arabidopsis thaliana has three RAD21 gene homologues, and their cloning and characterization are reported here. All three genes, AtRAD21.1, AtRAD21.2, and AtRAD21.3, are expressed in tissues rich in cells undergoing cell division, and AtRAD21.3 shows the highest relative level of expression. An increase in steady-state levels of AtRAD21.1 transcript was also observed, specifically after the induction of DNA damage. Phenotypic analysis of the atrad21.1 and atrad21.3 mutants revealed that neither of the single mutants was lethal, probably due to the redundancy in function of the AtRAD21 genes. However, AtRAD21.1 plays a critical role in recovery from DNA damage during seed imbibition, prior to germination, as atrad21.1 mutant seeds are hypersensitive to radiation damage.

Transposon activity is known to cause chromosome rearrangements in the host genome. Surprisingly, extremely little is known about Dissociation (Ds)-induced chromosome rearrangements in Arabidopsis, where Ds is intensively used for insertional mutagenesis. Here, we describe three Arabidopsis mutants with reduced fertility and propose that excision of a hybrid Ds element induced a large genomic deletion flanking Ds. In the mutants anat and haumea, the deletion mechanism consists of a local Ds transposition from replicated into unreplicated DNA followed by Ds excision, where one end of the newly transposed element and one end of the Ds transposon at the donor site served as substrate for transposase. Excision of this hybrid element reminiscent of a macrotransposon leads to loss of the chromosomal piece located between the two ends, including one full Ds element and the flanking genomic sequence. This mechanism was found to be responsible for several other deletions and occurs at a genetically trackable frequency. Thus, it could be applied to efficiently generate deletions of various sizes in the vicinity of any existing Ds element present in the genome. In the mutant tons missing, a mechanism that involves endogenous repetitive sequences caused a large flanking deletion at a position unlinked to the starter locus. Our study of Ds transposition in Arabidopsis revealed previously undescribed mechanisms that lead to large genomic deletions flanking Ds elements, which may contribute to genome dynamics and evolution.

Four recent papers have characterized the transcription profile of pollen grains, showing striking differences between gene expression in pollen and other plant tissues. These studies increase the number of known pollen-expressed genes by as much as 50-fold and have identified many novel genes that are potentially pollen-specific.