Genetic analysis of recC73 suppressor mutation mutant in Escherichia coli

U bakteriji Escherichia coli, enzim RecBCD zadužen je za obavljanje nekoliko važnih uloga u metabolizmu DNA. Enzim se sastoji od 3 podjedinice nazvane RecB, RecC i RecD koje zajedno čine holoenzim RecBCD. Svaka od podjedinica ima svoju karakterističnu ulogu, na temelju kojih holoenzim u cjelini može djelovati kao helikaza i nukleaza te imati sposobnost prepoznavanja Chi mjesta unutar DNA sekvence. Poznati su brojni mutanti enzima RecBCD koji ovisno o mjestu mutacije mijenjaju funkciju samog proteina pri čemu dolazi do promjene neke ili svih aktivnosti koje enzim obavlja. Mutant recC73 spada u takozvane null mutante jer je rekombinacijski deficijentan, nema nukleaznu aktivnost i ne može prepoznati Chi mjesto (Rec- Chi- Nuc-). Ustanovljeno je da mutacija recC73 predstavlja deleciju T na 1938 mjestu unutar gena recC uslijed čega dolazi do sinteze nekoliko pogrešnih aminokiselina i preuranjene terminacije translacije i sinteze krnjeg proteina RecC73. Supresorska mutacija, koja je nađena u promotorskoj regiji gena recC, je „poboljšavajuća“ mutacija koja uzrokuje pojačano prepisivanje ovog gena i povećanu količinu proteina RecC73. U ovom radu genetički je analizirana supresorska mutacija u mutantu recC73. Metodom qRT-PCR, pokazali smo da je količina transkripata za gen recC u suprimiranom mutantu recC73 povećana u originalni mutant recC73. Povećana ekspresija alela recC73 s plazmida mogla je suprimirati recC null mutaciju slično kao promotorska mutacija, dok povećana ekspresija ili gena recB ili recD s plazmida nije mogla suprimirati mutaciju recC73. Dobiveni rezultati potvrdili su da zbog povećane transkripcije alela recC73 može doći do djelomične supresije recC73 null mutacije te ukazuju da podjedinica RecC ima ključnu ulogu u sastavljanju enzima RecBCD.In Escherichia coli, RecBCD is responsible for performing a number of functions that include the DNA metabolism. The enzyme is composed of three subunits called RecB, RecD and RecC which together form a holoenzyme RecBCD. Each subunit has its own specific role, based on which enzyme as a whole can act as helicase and nuclease, and have the ability to recognize Chi sites within DNA sequences. Numerous mutants of RecBCD have been discovered, and depending on site of the mutation, roles of the enzyme can be altered in some or all of the activities that the enzyme performs. Mutant recC73 belongs to the so-called null mutants for it is recombination deficient, has no nuclease activity and cannot identify the location of Chi (Rec- Chi- Nuc-). Mutation recC73 is discovered to represent a deletion of T at the 1938 position within gene recC, causing a synthesis of several erroneous amino acids and premature termination of translation and synthesis of a truncated protein RecC73. Suppressor mutation, which is found in the promoter region of the gene is an "improving" mutation that causes increased transcription of the gene and an increased amount of protein RecC73. This work represents the genetic analysis of suppressor mutation in mutant recC73. By using qRT-PCR method, we showed that a certain amount of gene transcripts of recC in a suppressed mutant recC73 was increased when compared to the original mutant recC73. By overexpression of alleles recC73 from plasmids we achieved suppression of recC null mutations similar to the promoter mutation, whereas the effect of suppressing mutation recC73 through overexpression of genes recB or recD from plasmids has not been established. The results showed positive effect of suppression and the importance of the RecC subunit in the RecBCD assembly