Construction of E695A and Ile715M Mutants of

DNA polymerase I ITB1

 S. Arreneuz, F. Madayanti, R. Hertadi and Akhmaloka
Biochemistry Research Division, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung
Jl. Ganesha 10 Bandung, Indonesia, Email: loka@chem.itb.ac.id 

Abstract
DNA  polymerase  I  gene  from  one  of  local  thermophilic  microorganism  was  cloned  and  sequenced.  The  gene  has  been  expressed  in  E. coli
however structure function of the gene is being carried out. Computer prediction analysis of DNA Polymerase I ITB1  showed that E695 and 
I715 involved on the activity of the enzyme, especially on conformational change of DNA polymerase I during catalytic reaction. For further 
characterized the role of E695  and I715  on DNA polymerase activity, the E695A and I715M mutants were being constructed. All of the mutant 
genes were constructed by PCR mega primer. A few primers were designed and synthesized. The amplification of mutant genes were being 
carried  out.  Amplification  of  mega  primers  were  used  Pfu  DNA  polymerase,  meanwhile  amplification  at  the  whole  fragment  were  use  Taq
DNA  polymerase.  Both  mutants  were  amplification  and  confirmed  by  agarose  gel  electrophoresis.  Sub  cloning  at  the  mutant  fragment  to 
expression vector (pET30a) are still in progress..
Introduction Research Strategy
Computer Analysis

Construction of E695A and

Open Closed Open I715M
Mechanism of DNA Polymerase I for Nucleotide Selection (Patel, et al.,2001)

Structure Function Analysis of Mutant

Results
Result of computing analysis, E695 which hydrogren bonds with Serin residue and isoleusine residue have solvent accessible
surface area small. Design of primer for mutation E695A and I715M are primer of mutantE695A and I715M

A. DNA Pol I Wild Type B. DNA Pol I Mutant I715M

B. After mutation by simulation computer SASA become larger

Amplification of Gene Mutant Fragments

A. DNA Pol I Wild Type B. DNA Pol I Mutant E695A Mega primer  by Pfu The Whole genes by 
   A         B           P     1       2       λ        3        4
Taq
B. After mutation by simulation computer there was two hydrogen bonding
23130bp
9416bp
6557bp
Table Primer Mutant 1419bp
4361bp
Primer Urutan Primer Tm (0C) % GC % Homologi
RPBZ CTAGAGGATCCTTTATGTCGCGTCATACC 57.4 50 55 517bp 2322bp
PNCO 61.7 45.2 55 369bp 2027bp
Mutant E695A GACGATGACAAGGTACCCATGGAAAAAAAGC
AGGACGCAGTGACGCCCAACAT 58.6 59.1 59
Mutant I715M TTTGGGATCGTTTACGGGATGAGTGATTA 58.7 41.2 55

A. Megaprimer E695A 1 & 2 bands of gene E695A

Predicted B. Megaprimer I715M λ. λ/Hind III marker
P. pUC19/Hinf I marker 3 & 4 bands of gene I715M
 Glutamate and isoleusine involved within open and closed conformation change of DNA Polymerase I
 Mutation E695A cause to decrease hydrogen bond and mutation and its SASA mutation I715M become large
 Primer for mutant resulted two primer i.e. MutantE695A primer and MutantI715M primer

References
Patel, PH., Suzuki, M., Adman, E., Shinkai, A and Loeb, LA .,(2001), Prokaryotic DNA Polymerase I: Evolution, Structure,
and “Base Flipping” Mechanism for Nucleotide Selection (Review Article)

Meyer, AS., Blandino, M and Spratt, TE., (2004), Escherichia coli DNA Polymerase I (Klenow Fragment) Uses a Hydrogen-
bonding Fork from Arg668 to the Primer Terminus and Incoming Deoxynucleotide Triphosphate to Catalyze DNA Replication
(Accelerated Publication)