Aris Kaksis Riga University docent - Chemistry

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; GA

• GA

C 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'