A new type of E. coli recombinational hotspot which requires for activity both DNA replication termination events and the Chi sequence.

In E. coli rnh- mutants we identified chromosome-derived, specific DNA fragments termed Hot DNA. When the DNA in the ccc form is integrated into the E. coli genome by homologous recombination to form a directly repeated structure, a striking enhancement of excisional recombination between the repeats occurs. We obtained 8 groups of such Hot DNA, 7 of which were clustered in a narrow region called the replication terminus region (about 280 kb) on the circular E. coli genome. A Ter site can impede the replication fork in a polar fashion. The six Ter sites are approximately symmetrical in the terminus and surrounding region. To block the fork at the Ter site, a protein factor, Ter binding protein encoded in the tau (or tus) gene, is required. In tau- cells, Hot activity of HotA, B, and C DNAs disappears, thereby indicating that the Hot activity is fork arrest-dependent. Other Hot activities were tau-independent. In addition, for at least HotA activity, the presence of Chi, and E. coli recombinational hotspot sequence, is required; the Chi dependent HotA activity was detected in a wild type strain but to a lesser extent than that in the rnh- mutant. To explain the HotA phenomenon at the molecular level, we propose a model in which a ds-break occurs at the replication fork arrested at the Ter site. Our recent data that HOT1, a yeast recombinational hotspot, may also depend on the fork blocking event for activity, suggests that a similar ds-break occurs in both eucaryotes and procaryotes.