RNase-dependent discontinuities associated with the crossovers of spontaneously formed joint DNA molecules in Physarum polycephalum

Transient four stranded joint DNA molecules bridging sister chromatids constitute an intriguing feature of replicating genomes. Here, we studied their structure and frequency of formation in Physarum polycephalum. By “3D gels”, we evidenced that they are not made of four continuous DNA strands. Discontinuities, which do not interfere with the unique propensity of the joint DNA molecules to branch migrate in vitro, are linked to the crossover, enhanced by RNaseA, and affect at most half of the DNA strands. We propose a structural model of joint DNA molecules containing ribonucleotides inserted within one strand, a gapped strand, and two continuous DNA strands. We further show that spontaneous joint DNA molecules are short-lived and are as abundant as replication forks. Our results emphasize the highly frequent formation of joint DNA molecules involving newly replicated DNA in an untreated cell and uncover a transitory mechanism connecting the sister chromatids during S phase.