Chapter 7 --- DNA Replication

Read about the complications involved in trying to replicate double-stranded DNA

• The two strands are anti-parallel and this polarity has to be maintained
• Requires energy for unwinding in addition to the reactions of adding the nucleotides
• Single-stranded DNA will hybridize with itself if there is any complementarity
• There are a number of reactions involved, thus it must require an number of enzymes
• Replication errors occur, thus there have to be “correction” mechanisms
• Some DNA is circular, some is linear and very, very long, so there are major physical constraints on how this can proceed.

What is semi-conservative replication?

• See figures 7-1 and 7-2.
• Note the need for un-winding and then re-winding of the strands
• Remember that DNA polymerase only moves in one direction: 5’ à 3’

Replication of Circular DNA – theta replication -- q replication

• See figure 7-3 and 7-4
• Helicase is the basic un-winding enyme, but this introduces significant super-coiling effects
• Supercoiling is corrected by Topoisomerase
• Topoisomerase I – nicks one strand and relaxes the supercoil
• Topoisomerase II – Gyrase – cuts both strands

The picture you see is a three-dimensional reconstruction of a topoisomerase determined using data from X-ray crystallographic studies (blue, red, and yellow regions); from these data researchers can pinpoint the location of nearly every atom in the protein. The topoisomerase is modeled in the process of gripping a broken DNAduplex (shown in green) and transporting a second duplex (the multicolored rosette). This is part of the work carried out in the laboratory of JamesBerger in the Department of Molecular and Cell Biology .

Once thought to be mere laboratory curiosities, topoisomerases are now known to be essential for the livelihood of all organisms, from viruses and bacteria to humans. Moreover, it is now known that topoisomerases are targets for a large number of clinically used drugs, including anticancer agents and antibiotics. These drugs block the enzyme after it has cleaved the DNA, causing lethal breaks in the organism's chromosome. Understanding the molecular mechanisms behind the drug's action remains an important research goal, one in which the power of X-ray crystallography will play a critical role to resolve drug/topoisomerase interactions at atomic resolution.

Photo submitted by James Berger

• Replication starts at the Origin of Replication – Ori
• Two types of replication (Figure 7-6)
• De Novo – which needs primers (RNA primers)
• Rolling circle – Covalent Extension (Figure 7-9)

• De Novo Replication

• OriC (Chromosomal origin of replication) – is about 250 bp long – see fig 7-7
• It contains recognition sites for DNA binding proteins: Dna A and ATP
• This binding causes the denaturation of A+T rich sections of the ori and opens a “bubble”
• Unwinding in both directions is accomplished by helicase
• ssb – single-stranded DNA binding proteins – bind to the single strands and keep them from re-annealing
• RNA Primers are made:
• RNA polymerase on the leading strand
• RNA primase on the lagging strand
• DNA polymerase III extends the primer as DNA

· The polymerases:

o Polymerase I and II can be largely thought of as repair enzymes

o Polymerase III is the major replication enzyme

§ Require a template

§ Require dNTP’s

§ Require a “primer” which provides a free 3’-OH group to hook the next nucleotide onto

· Look over fig 7-11 to see the significance of a primer

· Note that synthesis occurs 5’ à 3’ (fig 7-12)

§ Both pol I and pol III have proofreading ability

· Called 3’ à 5’ exonuclease activity

· If wrong base inserted, i.e., no base-pairing after synthesis, the pol goes backwards and chews out the mis-matched base and tries again. (See fig 7-13)

§ pol I only: has 5’ à 3’ exonuclease activity: It can chew up any DNA that it bumps into AS it is synthesizing new DNA.

· This is important in a technique known as “nick-translation” (See fig. 7-14).

§ Compare the differences between pol I and pol III in table 7-1, page 131.

· The Mechanics of Replication

o Study figure 7-15 and note that this scheme is impossible because:

§ All the known DNA replicating enzymes use the 3’-OH group as the growing end. (The 5’-PO₄ end does not grow or elongate!)

o Instead, the scenario shown in figure 7-16 is believed to be the most common DNA replication mechanism. This is called the “discontinuous model”:

§ Lagging strand with many start sites. The direction of synthesis is away from the crotch of the replication fork.

· Many fragments called Okazaki fragments

§ Leading strand with one start site. The direction of synthesis is towards the crotch of the replication fork

o Pulse and pulse-chase experiments showed that new DNA consisted of two populations: Short and long, consistent with this model (fig 7-17 and below):

o Short pieces of RNA (1 to 60 bases long) serve as the primer for DNA synthesis. The primer is made by one of two enzymes:

§ RNA polymerase (rifampicin sensitive) makes the primer for the leading strand

§ Primase (rifampicin resistant) makes the primer for the lagging strand

· Primase forms a complex with the helicase – called the Primosome

o The complete picture:

§ Pol III continues to synthesize until it hits a previously made piece of RNA

§ Pol III drops off and Pol I takes over: since it has 5’à 3’ exonuclease activity, it can chop out the RNA primer until it gets to the DNA.

§ Pol I drops off and ligase seals the gap.

§ Note that the process is Bidirectional! See fig 7-22 and fig 7-24.

§ In E. coli, replication proceeds at 10⁵ nucleotides per minute!!! That is 1667 nucleotides per second!!!

What about Eucaryotes?

• Replication proceeds much more slowly: 500-5000 nucleotides per min, 8 – 80 nucleotides per second
• There are multiple initiation sites, multiple replication bubbles: See fig 7-25
• Also there is the histone “problem”: Histones need to dissociate from the DNA and then reassociate. Also need new synthesis of histones.
• Two different DNA polymerases:
• Pol a -- initiates lagging strand synthesis and also has primase activity
• Pol ∂ -- does the leading strand and completes the lagging strand fragments

Also there is the “telomerase problem”…

• Linear chromosomes will get shorter and shorter with each round of DNA replication
• The enzyme Telomerase solves this problem by extending the 3’ ends of the chromosomes with GC sequences. This is done without template.
• During replication, priming starts in this area, thus only the GC rich telomeres are lost

Finally, the book talks about AZT work --- What is his point??