Lecture 27 - DNA Replication PDF

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Summary

DNA replication...

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Word of the day… sequacious (adjective): following, imitating, or serving another person, especially in a reasonless manner.

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Pick up the worksheet from the front of the class. Get your clickers ready. This is a good time to form groups!

Some stuff to mention...  Targeted Tutorials  Extra office hours  New missed exam policy

Missed exam policy

DNA Replication

DNA Replication Learning Objectives  Compare and contrast DNA replication in vitro (PCR) and in vivo (in cells)  Explain the logistics of the DNA replication process including how the cell solves the problems of:  Separating the DNA strand.  Replicating both strands simultaneously in the 5’ to 3’ direction.  Synthesizing primers and providing primers for the leading and the lagging strands during replication (DNA synthesis).  Replicating the ends of the DNA.

DNA Replication Learning Objectives  Distinguish between, and label the leading and the lagging strands of DNA in a replication fork.  Explain what an Okazaki fragment is and its role in replication.  Explain how the ends of eukaryotic chromosomes are extended by telomerase, why this is necessary and important.  List examples of DNA-protein interactions during DNA replication (DNA Polymerase, helicase, RNA primase, DNA ligase, telomerase).  Predict the types of non-covalent interactions that enable interactions between DNA and DNA-binding proteins.

DNA Replication - Function  To make more of you!  DNA replication is the information for everything in the cell, so all daughter cells need it.  DNA replication in vivo (inside an organism) is therefore crucial!

DNA Polymerase  You saw this last lecture. The keys features are: • Reads a template DNA strand 3’ to 5’ • Makes the new complementary strand 5’ to 3’ • Requires a free 3’OH group on the end of an existing DNA strand (like a primer) to start.

Semi-conservative Replication  DNA replication is semi-conservative because each daughter cell receives one strand of DNA from the parent and the second that is freshly synthesized.

Which of the following are advantages of semi-conservative DNA replication? 1) If a base mismatch occurs in the DNA, only one of the daughter cells will be irreversible mutant. 2) If a full base pair is mutated (both bases changed), only one daughter cells will be a mutant. 3) It is more efficient than conservative replication (i.e. the two parent strands remain together).

A. 1, 2 B. 1, 3 C. 2, 3 D.1, 2, 3 E. Dunno

Which of the following are advantages of semi-conservative DNA replication? 1) If a base mismatch occurs in the DNA, only one of the daughter cells will be irreversible mutant. 2) If a full base pair is mutated (both bases changed), only one daughter cells will be a mutant. 3) It is more efficient than conservative replication (i.e. the two parent strands remain together).

A. 1, 2 B. 1, 3 C. 2, 3 D.1, 2, 3 E. Dunno

Semi-conservative Replication

DNA Polymerization

Replicating Circular DNA  Relatively straight forward biologically.  Replication starts at one place called the origin of replication (OriC) and continues until both directions meet up.

Replicating Linear DNA  Linear DNA tends to be huge and so multiple origin of replications (OriR) are used.

Rate of Replication  The rate of replication is about 1000 bps/sec. E. coli bacteria cell Genome = 4.6 million base pairs

Human cell - eukaryote Genome = 3.9 billion base pairs

Time to replicate entire genome with a single origin of replication

∼1 hour

∼45 days! But, only takes a few hours due to multiple OriR.

Activity time!

Worksheet - ANSWERS

b a

Primase

c

DNA Pol. I

Helicase

e f

g Topoisomerase

SSBPs

h

i DNA Pol. III

DNA Ligase

D

Extra Post-Class Notes  Just to emphasize, each of the two DNA strands can be the leading strand and lagging strand at the same time.  This is because each replication bubble has two forks that are moving away from each other.  In one direction either strand of the DNA is the leading strand for one fork, and the lagging strand for the other fork.

Replication Problems  The cell needs to solve three problems during the replication of DNA: 1) How to separate the DNA strands as needed. 2) How to make primers. 3) Allow synthesis to happen simultaneously in both directions on both strands.

Separating the Strands  It is impractical to separate the entire strands first within the cell, so the process needs to unwind just a bit at a time.  This requires two enzymes: helicase and topoisomerase.  Helicase unwinds the strands and topoisomerase relieves the stress caused by the helicase.

Making Primers  DNA polymerase needs a 3’OH to make DNA, therefore a DNA primer is impossible for the cell (a “chicken and the egg” problem).  The solution is provided by a special type of RNA polymerase called RNA primase.  Like all RNA polymerases, RNA primase does not need a primer in order to make RNA.  Thus, the cell uses RNA primers for DNA priming of replication.

Extra Post-Class Notes  RNA primase starts a new primer in a somewhat random way. The concentration of RNA primase in the cell makes it such that a new primer is made every 100-200 bases along the lagging strand.

Simultaneous Replication  Since DNA is anti-parallel and DNA polymerases only read 3’ to 5’, one direction of replication for both strands is in the wrong direction.  However, replication needs to go in both directions on both strands in order to be successful.

Simultaneous Replication  As the fork opens up, a gap forms on one strand (the same thing happens at the other fork).  The strand that replicates in a continuous direction is called the leading strand, while the other is the lagging strand.  Needs to fill in the gaps on the lagging strand!

GAP!

Simultaneous Replication  The gap is filled in by using another RNA primer and starting a new strand.  This results in a series of strand fragments called Okazaki fragments.

Simultaneous Replication  Eventually, all of the primers are replaced by DNA Polymerase I, and the nicks between the fragments are sealed up by DNA ligase....