RESEARCH SUMMARY

A central problem in biology is the faithful transmission of hereditary information from mother to daughter cells. This process not only involves precise replication of chromosomal DNA, but also correct partitioning of the newly synthesized sister chromosomes.

It has become clear over the last decade that the progression of replication forks in living cells and their viruses is disrupted with high frequency, by encountering various obstacles either on or in the DNA template. Survival of the organism then becomes dependent both on removal of the obstacle and on restart of DNA replication.

Homologous recombination is a process that takes place in all living cells to generate diversity, DNA repair, and for correct segregation of the chromosomes. It is also necessary to properly reassemble the arrested replication fork.

The study of homologous recombination mechanisms has revealed the complexity of the recombination process, due to the large number of proteins involved. Simple model systems such as bacteria and their viruses (bacteriophages) are therefore good candidates for deciphering these complex mechanisms.

We are analyzing how a replication fork is reassembled, and how replication can restart by a recombination-dependent mechanism, using Bacillus subtilis and its bacteriophage SPP1. In these two models, the outcome of the recombination-dependent replication is different, as in the bacteria the product is replication restart by a theta mechanism, whereas in SPP1, replication restart leads to concatemeric DNA synthesis which is the substrate for viral DNA packaging.

The latter type of replication is also found in herpes simplex virus, baculovirus, mitochondrial DNA, chloroplast DNA, telomeric circles, and certain pathogenicity islands, and takes place by a poorly characterized mechanism .

In the B. subtilis work, we focus mainly on the RecU Holliday junction (HJ) resolvase and the RecU modulators RuvA and RuvB. This enzyme has three activities:

(i) it cleaves HJ,

(ii) anneals complementary strands and

(iii) modulates RecA activities.

It interacts with the RecA recombinase and with the RuvB branch migration helicase. We have mapped the region essential for this interaction, and observed that RecU is recruited to the competence machinery, probably for modulating RecA activities at the competence pole.