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Identifier	000419740
Title	Identification and heterologous expression of CspAI restriction and modification system
Alternative Title	Ταυτοποίηση και ετερόλογη έκφραση του συστήματος για το περιοριστικό ένζυμο CspAI
Author	Μακρή, Ιωάννα Γ.
Thesis advisor	Κουτσιούλης, Δημήτρης
Reviewer	Κοκκινίδης, Μιχαήλ Αϊβαλιώτης, Μιχάλης
Abstract	Restriction endonucleases (REs) are enzymes which recognize and cleave a specific DNA sequence. Their biological function is to protect the host organism from invading DNA. In order to protect host’s genomic DNA, DNA modification enzymes are expressed, methyltransferases (MT). These enzymes methylate specific DNA sequences and protect them from respective REs cleavage. RE and MT coexist and compose a RM system. REs that recognize the same sequence but origin from different bacterial strains are called isoschizomers. Many isoschizomer pairs differ in their methyl-sensitivity. For example, CspAI and AgeI recognize sequence 5’-A/CCGGT-3’. When the underlined cytosine is methylated, CspAI cleave the DNA in contrast with AgeI that is blocked. Our ultimate goal is to compare for the first time the crystal structures of isoschizomers with different methyl sensitivity in order to identify the critical residues regarding methylation sensitivity. This data will help future studies on rational design of DNA modifying enzymes. Current thesis focuses on identification, cloning and expression of CspAI RM system components. Primarily, whole genome sequencing of Corynebacterium revealed the DNA sequences of R.CspAI and M.CspAI. Both genes were cloned and expressed in selected E.coli strain and R.CspAI expression was verified by western blot. Protein alignments of R.CspAI have demonstrated that it shares identical residues of the catalytic core with R.AgeI and hence, possesses the PD-(D/E)-XK motif. Further experiments will reveal if it has adapted the same cleavage mechanism with R.AgeI which is unique among Type IIP REs. M.CspAI sequence was compared against several MTs searching for conserved and characteristic motifs and showed higher similarities with N4-N6 MTs.
Language	English
Subject	R-M systems
Issue date	2018-11-23
Collection	School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
	Type of Work--Post-graduate theses
Permanent Link	https://elocus.lib.uoc.gr//dlib/a/2/1/metadata-dlib-1543661249-384460-3109.tkl
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