The role of Cas3 protein in R-loop formation in Escherichia coli

Sustav CRISPR/Cas nedavno je otkriveni novi način obrane bakterija od stranih genetičkih elemenata. Sastoji se od regije CRISPR (od eng. clustered regularly interspaced short palindromic repeat) i nekoliko gena cas (od eng. CRISPR-associated) u neposrednoj blizini. Otpornost se stječe ugradnjom kratkih fragmenata strane DNA unutar regije CRISPR, koji služe za prepoznavanje i cijepanje ciljne DNA prilikom ponovljene infekcije istim stranim elementom, slično RNAi kod eukariota. Korak u kojem dolazi do prepoznavanja i cijepanja strane DNA zove se interferencija, a u bakteriji Escherichia coli je poznato da je tu neophodan protein Cas3. U ovom radu istraženo je da li u koraku interferencije nastaje struktura nalik R-omči, tj. molekula RNA unutar dvolančane molekule DNA. To je istraženo praćenjem učinkovitosti transformacije i replikacije plazmida tipa ColE1 u kojem R-omča služi za početak replikacije. Također, praćen je utjecaj prekomjerne ekspresije proteina Cas3 na vijabilnost mutanata za koje je poznato da su uključeni u obradu R-omči. Dobiveni rezultati ukazuju da protein Cas3 stabilizira ili potiče nastanak R-omči.The bacterial CRISPR/Cas system is a recently discovered new defense mechanism against foreign genetic elements. It comprises a CRISPR region (clustered regularly interspaced short palindromic repeat) and a few adjacent cas (CRISPRassociated) genes. The resistance is acquired by integration of short foreign DNA sequences within the CRISPR region. These sequences then serve for recognition and cleavage of target DNA during recurring infection by foreign DNA, similar to RNAi in eukaryotes. The stage in which recognition and cleavage of foreign DNA takes place is known as interference, and protein Cas3 is indispensable for this stage in Escherichia coli. The purpose of this research was to test if a structure similar to R-loop, i.e. an RNA molecule hybridized within the DNA duplex, forms during the interference stage. To test this, the efficiency of transformation and replication of ColE1 type plasmids, which require R-loop formation for initiation of replication, has been studied.Furthermore, the effects of overexpressed Cas3 protein on viability of mutants, which are known to affect the R-loop formation, has been studied. Obtained results suggest that Cas3 protein stabilizes R-loops or promotes their formation