DNA Replication

Accel Bio 2014

Overview: What is DNA for?

• The purpose of DNA is to store the information necessary to allow cells & organisms to function. This includes storing all the info needed to make all of the proteins in our body.

• All of the DNA in a cell, which includes every gene, makes up an organism’s genome.

– The human genome is over 3 billion base pairs long!

• In our genome, the DNA is organized into structures called chromosomes.

What is a chromosome?• A chromosome consists

of a long piece of DNA wrapped up with proteins called histones.

• When DNA is wrapped with proteins, it is called chromatin.

• When a piece of chromatin is folded, coiled, & packed into a small bundle, it’s called a chromosome.• Humans have 46

chromosomes in every body cell.

ANIMATIONS: http://www.dnalc.org/resources/3d/07-how-dna-is-packaged-basic.html http://www.dnalc.org/resources/3d/08-how-dna-is-packaged-advanced.html

The Gene• The “functional unit” of DNA is the gene.• A GENE is the sequence of DNA that

contains the information required to make a specific _polypeptide_, from its first amino acid to its last.

• Questions:– How does the structure of DNA allow for

its function?1) How is DNA copied/replicated (without

errors!) when a cell divides?2) And, how does the process of making

a specific protein from a piece of DNA (a gene) work?

• The process of copying a double-stranded DNA molecule is called replication.

• Before cells can divide, DNA is replicated, usually without a single error! How??

• How difficult is this to do anyways? One way to think about it…

• Copying the 3 billion bases in human genome is like typing up a book that is 3 billion letters long.

• How many pages would this book be??– Assume average English word = 5 letters– Then, 3 billion bases = 600 million words– Assume 250 words / page (double-spaced 12 pt Times)– Then 600 million words = 2,400,000 PAGES– Could you type this book without any errors in 6-8

hours?

DNA Replication: a faithful process

• Copying the 3 billion bases in human genome is like typing up a book that is 3 billion letters long.

• How many pages would this book be??– Assume average English word = 5 letters– Then, 3 billion bases = 600 million words– Assume 250 words per page (double-spaced 12 pt Times)– Then 600 million words = 2,400,000 PAGES

• Could you type this book w/o any errors in 6-8 hrs?

• What strategies would help you accomplish this task??

DNA Replication: a faithful process

• How could we use one strand of DNA to know how to construct the second strand of DNA?

• The players: Enzymes like Helicase, Primase, DNA Polymerase, Ligase…

• The method: Complementary base pairing

DNA Replication

DNA Replication is Semi-Conservative

ANIMATION: http://www.dnalc.org/resources/3d/01-replication-the-helix.html

DNA Replication overviewBelow: DNA is copied quickly because there are several replication bubbles that open along a molecule at once. When the bubbles meet, replication is done.

One side of a replication bubble is called a replication fork (see below).

Steps of DNA Replication

1. The enzyme Helicase unwinds & “unzips” the DNA molecule at a particular point (an origin of replication), making two template strands of DNA available.

2. The enzyme Primase synthesizes (makes) short RNA primers (5’ 3’) to start each new DNA strand or fragment. The end of the primer is where DNA Polymerase begins.

3. The enzyme DNA Polymerase catalyzes the synthesis of the new DNA strands. DNA Polymerase helps the DNA nucleotides (which are readily available in the cell) match up with their complementary base on the template DNA (A & T, C & G).

4. DNA Polymerase also catalyzes the creation of covalent sugar-phosphate bonds along the backbone of the new DNA strands. (continued on next slide…)

Steps of DNA Replication, cont.

4. DNA Polymerase also catalyzes the creation of covalent sugar-phosphate bonds along the backbone of the new DNA strands.• DNA Polymerase creates the new strands of DNA in one

direction only -- from the 5’ end to the 3’ end of the NEW strand. This is because physically, DNA Polymerase can only add on to the 3’ end of the strand it is building.

• This results in one strand of DNA (the “leading” strand) being synthesized continuously from 5’ to 3’ while the other strand of DNA (the “lagging” strand) is synthesized from 5’ to 3’ in small pieces called Okazaki fragments.

• Why is one strand synthesized in small pieces? Because overall the strand needs to be made in the 3’ to 5’ direction, but DNA Polymerase cannot do this, so it makes several small 5’ to 3’ Okazaki fragments instead. Study the replication pictures to understand this better. Okazaki fragments are then joined together by yet another enzyme -- Ligase.

5. When synthesis of the two new strands is done, DNA Polymerase and other proofreading enzymes check for errors and repair them. Fantastic!

DNA Replication – the details

ANIMATIONS: http://www.dnalc.org/resources/3d/03-mechanism-of-replication-basic.html http://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html

Rates of DNA Replication

• How long does replication take?

• In the prokaryotic bacterium E. coli, replication can occur at a rate of 1,000 nucleotides per second.

• In comparison, eukaryotic human DNA replicates at a rate of 50 nucleotides per second.

• In both cases, replication occurs so quickly because multiple polymerases can synthesize two new strands at the same time by using each unwound strand from the original DNA double helix as a template.

http://www.nature.com/scitable/topicpage/cells-can-replicate-their-dna-precisely-6524830

DNA Replication Animations

• http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/dna-rna2.swf

• http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter3/animation__dna_replication__quiz_2_.html

• http://www.biostudio.com/d_%20DNA%20Replication%20Coordination%20Leading%20Lagging%20Strand%20Synthesis.htm

• http://www.johnkyrk.com/DNAreplication.html

• http://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html

A Labeled Replication Fork