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DNA Replication (Chapter 12)

Suggested problems: 1-3, 5, 7, 9-11, 13, 14, 18, 19, 21-24 see pp 350-351. Omit (now) recombination, pp 343-346.

Watson-Crick Watson-Crick model of model for Semiconservative Semiconservative DNA DNA replication replication

How does a double helix produce another double helix with precision?
5 It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material (Watson & Crick, 1953). 3 5 3 5 3

Three Hypotheses to explain DNA replication Supported by experiment

Semiconservative DNA replication


The two strands in the double helix separate, and then each strand serves as template for the synthesis of a new (complementary) strand. After replication has been completed, each of the two duplexes has one old and one newly synthesized strand. Conservative and dispersive modes of replication do not make much sense, and are not supported by experiments.

new old

Evidence for Semiconservative Replication


(See Fig. 12.2-12.3 p324-3255 in your text)

The Key Experiment


Grow a culture in 15NH4Cl and then transfer to 14NH4Cl. Allow cells to grow one generation (i.e., one DNA replication),
isolate DNA and subject to ultracentrifugation. Result: Semidense (15N/ 14N) DNA migrates to an intermediate position, providing evidence for semiconservative replication. Migration to an intermediate position suggests the presence of one heavy (15N) and one light (14N) chain in dsDNA.

Isolate DNA from E. coli cultures grown in 14NH4Cl (light) and


15NH 4 Cl

(heavy).

Subject DNA to ultracentrifugation in a density gradient of CsCl


solution. Result: Heavy (dense) DNA migrates faster than light DNA towards the bottom of centrifuge tube. Thus, heavy and light dsDNA molecules move to different positions in a density gradient and can be separated from each other.

The results (below) support semiconservative replication

Eukaryotic DNA replication is also semiconservative


Eukaryotic DNA also replicates semiconservatively as shown in Fava bean by the Taylor, Woods, and Hughes experiment in 1958. They labeled DNA with 3H-T, treated the roots with Colchicin, fixed and prepared for microscopy. At the first metaphase, after labeling at interphase, both chromatids of each chromosome were labeled, whereas at the second metaphase only one chromatid was labeled. How does this show semiconservative replication? See next slide

heavy

mixed

light

because they each have a newly synthesized (labeled, light blue) DNA chain

Other features of DNA replication


Bidirectional. Starts at specific sites (origins) and moves in opposite directions using two replication forks. Semi-discontinuous. One strand (leading) replicates continuously and the other (lagging) discontinuously (Fig. 12.10, p331). In the 5 - 3 direction. Enzymes (DNA polymerases) can only add a nucleotide to a free OH group at the 3'-end of a growing chain (Fig. 12.7-8, p329-330).

A replication Fork

Each fork at a replicon has a lagging strand and a leading strand

One enzyme dimer synthesizes both the leading and the lagging strand

DNA replication requires:


Template (ssDNA) Primer (RNA) Substrates (dNTPs: dATP, dGTP, dCTP, dTTP) Enzymes (helicase, topoisomerase, polymerases, primase, ligase, and other protein factors)

Other aspects of replication :


DNA replication begins at a specific site or sites called origin. The origin is recognized by specific initiator proteins. Viruses. Circular DNA, one origin, rolling circle, unidirectional (one fork). (Fig. 12.5, p327). Prokaryotes. Circular DNA, one origin, theta scheme, at a rate of 1,000 nt per second (20-40 min) (Fig. 12.4, p326). Eukaryotes. Linear DNA, multiple (500-25,000) origins at a rate of 10-50 nt per second (3-4 min to hours) (Fig. 12.6, p328).

Replication of prokaryotic DNA --Theta model

Replicon

(Unidirectional Replication)

Replication of linear DNA using multiple origins

Multiple origins of replication in eukaryotic DNA

Steps in DNA Replication


1. Initiation and unwinding

Binding of initiator proteins to the origin and strand


separation (melting) by helicase*

ssDNA strand stabilization by ssDNA-binding proteins Torsional strain release by gyrase or topoisomerase Priming (primase: a special RNA polymerase; Pol in
eukaryotes) When all of the above factors are in place, DNA polymerase can bind to the template and start synthesis. *Bloom syndrome is due to a mutant helicase.

Each origin produces a replicon with two forks, and the forks move in opposite direction Initiation of Replication at an origin

Left Fork

Right Fork

Steps in DNA Replication, cont.


2. Elongation
Addition of dNTPs (chain elongation by DNA pol III starting at the 3-end of the RNA primer. Thus, the chain grows in the 5-3 direction) Proofreading (DNA polymerase III, 3-5 exonuclease ) Excision of RNA primer + gap filling (Polymerase I removes RNA nucleotide and replaces it with DNA nucleotide, 5-3 exonuclease) Splicing of fragments (DNA ligase links the Okazaki fragments after Pol I fills the gap)

5 to 3 chain elongation by adding new nucleotides to the 3-end of the growing chain

3. Termination occurs when two forks meet each other or


a specific termination signal is encountered.

RNA Primer is extended with DNA

DNA fragments (Okazaki fragments) are spliced (ligated)

RNA Primer is removed and replaced with DNA

A misincorporated base (A in this example) is removed and then replaced with a correct base (C in this example).

Mismatch repair corrects a wrong base in newly replicated DNA. The newly replicated DNA is recognized based on its lack of methylation

Additional Points
There are 2 DNA polymerases (I and III) in prokaryotes

Additional Points (cont.)


Not surprising, DNA replication has extremely high

as opposed to 4 (, , , ) in eukaryotes. Okazaki fragments are synthesized by Pol III, , , . Organelle DNA is synthesized by . There are other DNA polymerases; they are involved in DNA repair.

fidelity as a result DNA polymerases accuracy, proofreading activity, and post-replication mismatch repair.

The same enzyme molecule (a dimer) synthesizes both the leading and the lagging strand. The enzyme has a fixed position within the nucleus, the DNA template is threaded through it. The telomeres or the ends of eukaryotic chromosomes are synthesized by a special reverse transcriptase (telomerase). Lack of telomerase activity leads to shortening of chromosome ends and is implicated in aging and eventually death. Cancer cells do not lose telomerase activity.

In eukaryotes, many replication origins are needed, and they are used only once under the control of licensing proteins (binding of licensing proteins). What would be the result without licensing? Prior to replication, the nucleosome must dissociate (histones removed) to expose the naked DNA to the replication machinery. After replication, the nucleosome reassembles, which requires doubling the amount of histones.

has a gap

Telomerase brings an RNA template and lls the gap

This gap cannot be lled by DNA polymerases because they cannot extend a chain in the 3-5 direction

Further extension of the chromosome end by telomerase

The chain extended by telomerase loops back, serves as a primer, and its 3end is extended by DNA polymerase

DNA Synthesis and Hydrolysis Enzymes


DNA-dependent DNA polymerases Require template and primer. Extend primer in the 5 - 3 direction. Example: Pol III, Pol . DNA-dependent RNA polymerases Require template but not primer. Synthesize RNA in the 5 - 3 direction. Exonucleases 5 - 3 exonucleases: digest DNA or RNA in the 5 - 3 direction (Pol I). 3 - 5 exonucleases: digest in the 3 - 5 (Pol III). Endonucleases Cleaves phosphodiester bonds internally. 1. Non-specific (DNases; RNases). 2. Specific (Restriction endonucleases)

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