Instructor: Dr. Natalia Tretyakova, Ph.D. «hyperlink "mailto:Trety001@umn.edu"»    -   6-3432
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Prof. e-mail: :ariska@latnet.lv
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           
Template

 

Figure 1. Two – metal – ion mechanism.

 

E. Coli DNA Polymerases
 
Characteristic                      Pol I                      Pol II             Pol III

Mol. Weight (Da)                 103,000                 88,000            900,000
Number of polypeptides       1                            4                     10
rate (nucleotides/sec)           16-20                     7                     250-1000
 3'
--> 5' exonuclease             yes                         yes                 yes
 5'
--> 3' exonuclease             yes                         no                   no
function                                Primer removal,    unknown        Major
                                              gap filling                                    replicative polymerase

 
DNA Polymerization Has Three Stages
 
1) Initiation
 
2) Priming
 
3) Processive Synthesis

 

DNA Polymerization: Initiation
 
        DNA replication begins at a specific site.
 
       Example: oriC site from E. coli.
          • 245 bp out of 4,000,000 bp
          • contains tandem array of three 13-mers; GACNN 
          GAC common motif in oriC
          A bp are common to facilitate unwinding

 

Initiation of DNA Polymerization
 
         Tandem Array
   (13-Mer Seq. A- Rich)                                                       dna A

                     Ori C                                         dna A protein unwinds oriC  at several sites
 
Enzymes involved in the initiation of DNA Polymerization
 
Enzyme                         Function
 
dna A                            recognize replication origin and         unwinds it at several sites
 
Helicase (dnaB)            unwinding of ds DNA
 
DNA gyrase                  generates (-) supercoiling that compensates for the (+)
                                      supercoiling generated in front of the replication fork
 
SSB                               stabilize unwound ssDNA
 
Primase                        a RNA polymerase that generates RNA primer.
 
Priming of DNA Polymerization  Primer Synthesis and removal
 
3'  5'
|| Primase
3'  5'
5'  3'
RNA primer || DNA Pol III
3'  5' 
5' ---->       3' 
 || DNA Pol I
DNA strand                           3'  5' 
5'  3' 
degraded               <-- primer + || DNA Ligase
3'  5' 
5'  3' 
 
DNA Polymerization
 
Processive Synthesis
The synthesis of DNA in E. Coli is carried out by the DNA polymerase III holoenzyme.
 
• Extremely high processivity; once it combines with the DNA and starts it                                                                                           does not come off until finished.
• Tremendous catalytic potential; 1000 nucleotides per sec.
• Low error rate (high fidelity) 1 error per 10,000,000,000
 
alpha subunit is the polymerase
epsilon subunit is the 3'--> 5' exonuclease
beta subunit provides a tight clamp for sliding        DNA through
 
            Pol III
 
         Pol III                                              Pol III + DNA
 

                     ½¾ 50 Å ¾ ¾ ½
                                          DNA SynthesisDNA Synthesis
                                                                   5'       3'

 3'       5'                                                                                                                       3'         5'