The effect of point mutations on the activity and secondary structure of Cas3 protein in Escherichia coli

Sustav CRISPR-Cas predstavlja važan mehanizam obrane prokariota od stranih molekula DNA i RNA. Sastoji se od lokusa CRISPR, kojeg čine ponavljajuće sekvence DNA između kojih se nalaze razmaknice porijeklom iz plazmida ili bakteriofaga, i geni cas. Transkripcijom lokusa CRISPR nastaje pre-crRNA iz koje procesiranjem nastaju crRNA komplementarne stranoj DNA ili RNA. Udruživanjem s proteinskim kompleksom proteina Cas, crRNA služe kao smjernica za prepoznavanje stranih nukleinskih kiselina. Kompleks Cascade dovodi protein Cas3 koji potom degradira ciljnu DNA. Cas3 sadrži nukleaznu i helikaznu domenu čijom aktivnošću štiti bakteriju od invazije stranom DNA. Aktivnost Cas3 ovisna je o temperaturi i primijećeno je da pri temperaturi od 35 °C dolazi do suptilne promjene u helikoidalnoj regiji pročišćenog proteina Cas3 te gubitka zaštitne aktivnosti sustava CRISPR-Cas in vivo. U ovom radu cilj je bio uvođenjem točkastih mutacija u tri različite -zavojnice proteina Cas3 pronaći onu koja mijenja položaj i time utječe na aktivnost proteina. Praćenjem promjene sekundarne strukture proteina Cas3 u ovisnosti o temperaturi in vitro utvrđeno je da dvije -zavojnice nisu odgovorne za gubitak aktivnosti. Iz preživljenja faga λ rezultati ukazuju na mogućnost da bi -zavojnica smještena najbliže nukleaznom centru mogla biti odgovorna za promjenu aktivnosti proteina Cas3, a uočena je samo u proteinu Cas3 bakterije Escherichia coli.The CRISPR-Cas system represents an important defense mechanism of prokaryotes against mobile genetic elements. It is composed of an array of short DNA repeats separated by spacer sequences derived from plasmids or bacteriophages, and cas genes. Pre-crRNA is transcribed from the CRISPR array and is processed into short crRNAs. Incorporated into protein complex, crRNA serves as a guide for foreign nucleic acid recognition. Protein complex then recruits Cas3 protein that degrades the target DNA. Cas3 consists of nuclease and helicase domain whose activities protect bacteria from invading DNA. Cas3 protein activity is temperature dependent, and it was observed that at 35 °C subtle changes occurred in the helical region of the purified Cas3 and protection by the CRISPR-Cas activity was lost in vivo. The aim of this work was to introduce point mutations into three different -helixes of the Cas3 protein and identify the one responsible for altering position and thus affecting the protein activity. By measuring the changes in secondary structure of the Cas3 protein at different temperatures in vitro, it was found that two -helixes are not responsible for the loss of the Cas3 activity. In vivo results suggest that -helix situated near the nuclease centre may be responsible for the change in Cas3 activity, and it is observed only in Cas3 in Escherichia coli