DNA Watson and Crick’s hypothesis had immediately suggested a possible self-replication method for DNA because of complementarity in base pairing.
At this junction, possible modes of DNA replication: Conservative / Semi-conservative / Dispersive
- Meselson-Stahl Experiment proves replication is semi-conservative
Bacteria grown in N(15) medium incorporate N14 in a light medium - CsCl density gradient centrifugation revealed.
- Identification of Replication Fork
John Cairns identified the replication fork by allowing replicating DNA in cells to incorporate tritiated thymidine. Each daughter molecule contains a radioactive strand and a cold strand.
- Is replication bidirectional?
Gyurasits and Wake - Replication bubble lightly labeled first, and placed in medium with higher radioactivity. Bubble has heavily labeled fragments at both ends.
- Origin of replication
An origin of replication is a sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. For small DNAs, including bacterial plasmids and small viruses, a single origin is sufficient. Larger DNAs have many origins, and DNA replication is initiated at all of them; otherwise, if all replication had to proceed from a single origin, it would take too long to replicate the entire DNA.
- Enzymes for DNA replication
A. DNA polymerase - Required for elongation of DNA strands. Needs a primer and a template. (only in vivo)
Q. What is a primer?
A primer is a strand segment with a free 3’OH group, to which a nucleotide can be added (primer terminus).
Q. Why is RNA chosen as primer instead of DNA?
DNA pols can only add to a 3’OH GROUP and CANNOT initiate replication on its own. RNAses can. Voila.
Q. What is the consequence of incorrect base pairing on catalysis by DNA polymerase?
The incorrect base pairing mostly hinders further action by the DNA polymerase, and the incorrect alignment reduces the rate of catalysis dramatically. SO the DNA pol preferentially adds correct bases.
Q. How does the DNA polymerase ensure high fidelity during replication?
Most of the mistakes during DNA replication are promptly corrected by DNA polymerase by proofreading the base that has been just added (Figure 14.6.1). In proofreading, the DNA pol reads the newly added base before adding the next one, so a correction can be made. The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the wrong nucleotide. This is performed by the exonuclease action of DNA pol III. Once the incorrect nucleotide has been removed, a new one will be added again.
Q. What are Okazaki fragments?
One strand, which is complementary to the 3’ to 5’ parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5’ to 3’ parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand.
Q. What is the benefit of the looping of the lagging strand along the replisome?
The looping back of the lagging strand onto the replisome allows both leading- and lagging-strand DNA polymerases to synthesize in the same direction and facilitates recycling of the lagging-strand DNA polymerase by virtue of its proximity to the RNA primers newly synthesized at the fork.