DNA Replication

Part 2Enzymology

Figure 11.10

The Polymerization Reaction

Main E. coli DNA Polymerases• DNA polymerase I

– Single subunit enzyme – Degrades RNA primers and fills gaps– DNA repair– 5'3' polymerase activity– 3’5’ exonuclease activity – proofreading function– 5’3’ exonuclease activity – primer removal & repair function

• DNA polymerase III– Main DNA polymerase for replication of genome and plasmids– Multiple subunit holoenzyme – different subunits for pol and exo

activities– 5'3' polymerase activity– 3’5’ exonuclease activity – proofreading function

Eukaryotic DNA Polymerases

Catalytic domain of E. coli DNA Polymerase I

holoenzyme

Pol III*

E. coli DNA Pol III Holoenzyme

Sliding ClampsE. Coli dimer clamp Human trimer clamp

Subunit PCNA

Enzymology of DNA Synthesis

• DNA-dependent DNA Polymerases– require a DNA template

• Require a free 3'-OH– a primer

• NTP added to 3'-OH of growing polynucleotide chain– 5' 3' synthesis

Mechanics of DNA Synthesis

Architecture of the Replisome

535

3

35

5

HelicaseTopoisomerase

ssDNA-bindingproteins

DNApolymerase RNA

primer

Leading strand

Lagging strand RNAprimer

Primase

DNA Pol I Finishes the Job

• DNA Pol I has 5'3' exonuclease function that destroys RNA primer

• As RNA is removed, DNA is replaced by the 5'3' polymerase function

• DNA ligase seals the gap by joining the 3'OH to the 5'PO4

Termination of Replication: Circular Chromosomes

Figure 11.24

What happens at the end of a linear chromosome?• The need for a primer poses a problem at the 3’

end of linear chromosomes

• Linear eukaryotic chromosomes have telomeres at both ends

• The term telomere refers to the complex of telomeric DNA sequences and bound proteins

Telomeres and DNA Replication

Figure 11.23

• Telomeric sequences consist of hexanucleotide or heptanucleotide repeats

• The very end of telomeres are a 12-16 nucleotides 3’ overhang formed by removal of the RNA primer after DNA synthesis

Telomere Sequence & Structure

Replication of Telomere Sequences• In mammalian somatic cells, 100-1000 telomere

repeats are lost with each round of replication• Telomere sequence must be maintained in gamete

precursors– Done by telomerase

• Telomerase– utilized a RNA template to extent the 3’ end of one strand

at the telomere– telomerase has reverse transcriptase enzyme activity– The RNA is complementary to the DNA sequence found in

the telomeric repeat

Figure 11.25

Step 1 = Binding

Step 3 = Translocation

The complementarystrand is made by primase, DNA polymerase and ligase

RNA primer

Step 2 = PolymerizationReverse transcriptase

Telomerase & Telomere Extension

Telomeres Protect Ends of Chromosomes