DNA Replication

Part 2

Enzymology

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 Clamps

E. 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