|
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
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School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
|
|
Type of Work--Post-graduate theses
|
Views |
334 |