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DNA replication is essential for the propagation of life and the development of complex organisms. However, replication is a risky process as it can lead to mutations and chromosomal alterations. Conditions challenging DNA synthesis by replicative polymerases or DNA helix unwinding, generally termed as replication stress, can halt replication fork progression. Stalled replication forks are unstable, and mechanisms exist to protect their integrity, which promote an efficient restart of DNA synthesis and counteract fork collapse characterized by the accumulation of DNA lesions and mutagenic events. DNA replication is a highly regulated process, and several mechanisms control replication timing and integrity both during unperturbed cell cycles and in response to replication stress. Work over the last two decades has revealed that key steps of DNA replication are controlled by conjugation of the small peptide ubiquitin. While ubiquitylation was traditionally linked to protein degradation, the complexity and flexibility of the ubiquitin system in regulating protein function have recently emerged. Here we review the multiple roles exerted by ubiquitin-conjugating enzymes and ubiquitin-specific proteases, as well as readers of ubiquitin chains, in the control of eukaryotic DNA replication and replication-coupled DNA damage tolerance and repair.

Three different blood units were treated separately by the hypotonic dialysis (HD) and the dimethylsulphoxide osmotic pulse (DMSO) method, in order to load the erythrocytes with inositol hexaphosphate. A detailed comparison between the two loading techniques was performed by monitoring the red cell distribution patterns on discontinuous Percoll density gradients, the RBC oxygen affinity and the amount of the main intracellular organic phosphates with the 31P-NMR. The results obtained showed that: (1) The HD loading produces a redistribution of the RBC fractions with a concomitant smoothing of the relative differences among distinct fractions (2) only a minor portion of erythrocytes (from 8.5 to 24.9% of total RBCs) are loaded with IHP after the DMSO treatment. All of these cells move to the lightest fraction (d = 1.080 g/ml). (3) Both HD and DMSO IHP-loaded cells show an increase in P50 (basal vs. after loading, means +/- SD: 25.8 +/- 3.0 vs. 52.5 +/- 3.2 mm Hg) correlated to the IHP incorporation (mean intracellular IHP concentration: 4.2 mmol/l RBC). (4) probably the IHP incorporation efficiency could be probably improved at least by increasing the IHP concentration during the treatment.