DNA REPLICATION - National University replication.pdf · 2019-03-19 · DNA Replication Main...
Transcript of DNA REPLICATION - National University replication.pdf · 2019-03-19 · DNA Replication Main...
Ashraf Yahia Osman Mohamed MBBS (UofK), MSc (IEND-UofK) Whatsapp:00249922577404
DNA REPLICATION
Why DNA replicates???
DNA ReplicationMain classes of enzymes and proteins involved in DNA synthesis DNA Polymerase
Addition of nucleotides, repair and removal of primers
Helicase Opening of DNA helix by breaking hydrogen bonds
Topoisomerase Relieve the stress on DNA caused by unwinding by breaking and resealing the DNA breaks
Ligase Seals DNA gaps Primase Synthesis primers Single Strand Binding Proteins (SSB)
Bind single strands of DNA to prevent DNA from rewinding back
DNA Replication
Stages of DNA replication• Initiation
• Elongation
• Termination
DNA Replication
Where??
When??
How??
At which specifc Nucleotide should replication start??
Initiation
• For replication to begin the DNA strands have to be separated from each other.
• Strand separation is done by DNA helicase.
Replicator
HelicaseLoading protein
Initiator
Initiation
Main proteins involved in initiation
Protein E. coli (prokaryotes) Yeasts (eukaryotes)
Replicator oriC Autonomously replicating sequences (AUR)
Initiator DNaA Origin recognition complex (ORC)
Helicase DNaB Minichromsome maintenance complex (MCM)
Loading protein DNaC Cdc6 & cdt1
SSB SSB Replication protein A (RPA)
Initiation
Steps of replication initiation• Replicator recognition
• Helicase loading
• DNA unwinding (re-annealing is prevented by single strand binding proteins SSB).
Initiation
• Eukaryotic chromosomes are generally larger than prokaryotic chromosomes this why multiple replication origins are required.
Elongation
• The old DNA strands act as templates for synthesis of the new duplex.
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Elongation
• DNA replication is semi-conservative• DNA replication is semi-conservative
Elongation
• New DNA strands are synthesized by the enzyme DNA polymerase
• Unwinding is proceeded by helicase and re-annealing is prevented by SSB.
• Topological stress is relieved by topoisomerase
Elongation
• DNA polymerase requires RNA primer to start the synthesis.
• A primer is an oligonucleotide (most commonly of RNA) complementary to the template to which an enzyme add additional nucleotides.
• Primers are synthesized by the enzyme primase.
Elongation
Figure DNA polymerase reaction
Elongation
• DNA synthesis is bidirectional and proceed in the 5’-> 3’ direction.
• DNA synthesis is bidirectional and proceed in the 5’-> 3’ direction.
Elongation
Elongation
Elongation
• DNA replication begins at an origin of replication and proceed in a semi-discontinuous manner.
• DNA replication begins at an origin of replication and proceed in a semi-discontinuous manner.
Elongation
• The leading strand requires only one priming event (which occur at the origin)
• The lagging strand requires a series of priming events since each Okazaki fragment requires its own start.
Elongation
• There are several types of DNA polymerases.
● Not all polymerases are good DNA replicators
● In E. coli the most important polymerase is polymeraseIII while in humans the most important are DNA polymerase ε and δ.
Elongation
Processivity • Number of nucleotides added before
the enzyme dissociates.• Processivity of polymeraseIII is
enhanced by β subunits.• Processivity of polymerase δ is
enhanced by binding to proliferating cell nuclear antigen (PCNA).
Elongation
Elongation
Fidelity • The accuracy of the polymerization.• Fidelity is enhanced by the 3’->5’
exonuclease proof-reading activity of the polymerase.
Elongation
Elongation
• Some DNA polymerases have functions beside the replicative polymerization.
e.g: 5->3 exonuclease activity of DNA polymerase I which removes the primers and have a role in DNA repair.
Elongation
• Primers are removed by specifc DNA polymerase I.
• Nicks are sealed by Ligases.
Elongation
• Some DNA polymerases have non-replicative functions.
e.g: The reparative polymerization done by eukaryotic polymerase η.
Termination
• DNA replication is terminated after interaction between the converging replication forks.
Termination
• Specifc sequence in the bacterial DNA (known as Ter) binds to a protein known as Tus.
• The Ter-Tus complex halts one replication fork until it collides with the other.
Suggested Readings: Harper's Biochemistry
Chapter of DNA organization, replication and repair• DNA synthesis Lehninger Principles of Biochemistry
Chapter 25• DNA Replication Lippincott’s Biochemistry
Unit 6• Steps in Prokaryotic DNA Synthesis• Eukaryotic DNA Replication