Rules of Replication

1. Semi-conservative
2. Primer is required
3. Template is required
4. Elongation occurs 5' -> 3' (Template is read 3' -> 5')
5. Proof reading is done by 3' -> 5' exonuclease
6. Semi-discontinuous
7. Bidirectional

Some more details about DNA Polymerase III

A complex enzyme with many subunits:

Alpha subunit is the core synthesis part.

Epsilon subunit is responsible for proofreading

Beta subunit is the "sliding clamp" that keeps the enzyme associated with DNA

(There are several more subunits (theta, delta, gamma, etc) but for the most part you only need to think about the first three).

Each of those subunits is coded by a different gene

Fig. 3.8 Model for the "replication machine," or replisome, the complex of key
replication proteins, with the DNA at the replication fork

This figure should be familiar. Note that our current understanding is that the replication "factory" is stationary and the DNA moves through the enzyme.

Why does one strand make a loop?

You should review the animations on your textbook CD

You may also want to look at the animations and interactive problems at

http://www.dnai.org

(go to the section on "copying the code")

Proofreading

Even though bases preferentially pair G-C and A-T, the error rate is about 1 in 10,000.

Many polymerases have "proofreading" ability. They can excise an unpaired base (3' ->5' exonuclease activity)

This reduces the error rate to about 1 in a billion.

How does it work?

Incorrect pairing probably stalls the replication fork, allowing the unpaired nucleotide to migrate into the exonuclease site in the polymerase complex.

(see 3-d picture of polymerase complex)

Eukaryotic DNA replication is similar, but more so

At least 5 polymerases in mammals

(a, b,d, g,e). Some nuclear, some mitochondrial, some show proofreading, some not.

DNA synthesis is 10x slower (only 50 nt/sec)

There are multiple origins of replication

Necessary because much more DNA in Eukaryotes.

Need a mechanism to license the replication origins to ensure only one copy is made each cycle

New DNA has to be assembled into nucleosomes

What happens to the lagging strand that the end of the chromosome?

Removal of RNA primer leaves a gap at the 3' end

Convince yourself that this is true and that it is a problem.

Removal of the gap requires a special enzyme (telomerase) to add additional sequence to the 3' end. The enzyme is a mix of protein and RNA template

Telomeres contain hundreds of simple tandem repeats.

In humans, the repeat sequence is TTAGGG

Cell lines with active telomerase live longer than those without telomerase. That may be important in allowing cancer cells to continue to divide.

Study figs 3.18 and 3.19 in your book to see the action of telomerase

Chapter 3 problems

2, 4, 8, 13, 20, 21, 23, 25

More questions to organize your studying:>

* How is the origin of replication recognized?

* How are the two strands melted?

* How are the strands kept apart?

* What enzyme makes the RNA primer?

* What enzyme removes the RNA primer?

* What unwinds the double helix at the replication fork?

* How is the torsional stress caused by unwinding relieved?

* Is there any difference in synthesis of the leading and the lagging strand?

* What joins the Okazaki fragments together?

* How is replication terminated?

* How are new chromosomes separated?

* How is all DNA synthesis coordinated with cell division?