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Lecture 5

Lecture 5 Hour 1

Course Code
Jane Mitchell

of 3
Lecture 5 (Yip)
Feb. 2/12
Introduction to DNA Replication (2)
Replication direction
Newly synthesized strand is synthesized from 5’-3’
The 5’ of the complementary strand attaches to the 3’ of the parent
Replication origin
Usually starts at same recognized replication sites by initiator proteins, non-random
Site must be A-T rich (2H bonds v. 3H bonds for G-C), easier to open
Single replication origin at prokaryotic bacteria
Multiple replication origins for eukaryotes
Origin identification experiment (circular genome, bacteria)
Use ARS (autonomously replicating sequences) in a yeast cell
ARS functions as an origin of replication
Compare two yeast cells with a common gene (ex. His gene)
Insert possible ARS segment in one and random DNA segment in the other
Replication and common gene expression should be greater in ARS induced cell
DNA replication (circular genome, bacteria)
Proteins attach to single replication origin
Pull double stranded circular genome apart
Replication occurs at replications forks
Bi-directional growth from one starting point (left and right)
Forks are points where parental strands are pulled apart
Replication at forks
Obeys two rules:
1) DNA strands are anti-parallel
2) Newly synthesized end adds on to 3’ end of parent strand
Lecture 5 (Yip)
Feb. 2/12
Parental strands divided into leading and lagging template
Leading template runs from 3’-5’ (5’ at replication fork)
Leading strand synthesizes as a continuous strand from its 5’-3’
Lagging template runs from 5’-3’ (because DNA is anti-parallel)
Lagging strand (Okazaki fragments) use a fragmented “back-stitching” method
Okazaki fragments due this in order to be anti-parallel to lagging template
Replication steps (circular genome, bacteria)
1) Origin of replication is identified
2) Initiator proteins bind to replication cite
Helps helicase bind to DNA
Requires ATP
3) Unwinding by helicase
Helicase unwinds and separates the two parental strands
Two types of helicase
Predominant helicase moves along lagging template in 5’-3’ direction
4) Binding of single-strand binding proteins (SSB proteins)
Bind to DNA template to prevent re-annealing (prevent strands from re-forming H-
Found on lagging strand to improve back-stitching mechanism
5) Introduction of RNA primers
RNA primer is simply a single stranded complementary nucleotide sequence
Primer serves as starting point of DNA synthesis between complementary/template
DNA primase (protein) creates RNA primer in 5’-3’ direction
RNA primer is removed and replaced later
Located on the lagging strand
DNA primase + DNA helicase = primosome
6) DNA polymerase
Active after the introduction of an RNA primer
Enzyme that catalyzes the synthesis of nucleotides
Catalyzes incoming dNTPs (deoxyribonucleotide triphosphate)
Removes pyrophosphate to form a nucleotide
Works in 5’-3’
Lecture 5 (Yip)
Feb. 2/12
Found on both strands
7) Sliding clamp holds polymerase onto DNA
Circular protein that attaches itself to DNA polymerase
Prevents DNA polymerase from falling off the DNA strands during replication
Found on both strands
8) Nick sealing by DNA ligase
Active after RNA primer on Okazaki fragments is removed
DNA ligase joins new Okazaki fragments into a growing chain