Question

A single replisome complex can simultaneously synthesize both the leading and lagging strands of DNA within a moving replication

fork.

(a) Why is this challenging?

(b) Use the “trombone model” analogy to explain how a single replisome synthesizes the lagging strand at the same time the

leading strand is being made.

Answer 1

(a)

DNA is a double helical molecule with anti parallel strands which are complementary to each other.

5'- ATGCGATCGA -3'

3'- TACGCTAGCT -5'

DNA polymerase is an enzyme which synthesizes DNA. In E. coli DNA polymerase III is the main replicating polymerase. It synthesizes DNA in the 3' to 6' direction using 5' to 3' as the template. It requires a primer which provides a 3'-OH group which the enzyme extends and goes on adding numcleotides as part of its polymerase activity guided by the base sequence of the template.

Now, the DNA strand with polarity 5' to 3' is called the leading strand while the other is the lagging strand. On the leading strand, continous DNA synthesis occurs while on the lagging strand discontinous DNA synthesis occurs resulting in the formation of OkaZaki fragments.

The problem in synthesizing both strands simultaneously is that the polymerization occurs in 3' to 5' direction and polymerase moves in 3' to 5' direction but the the two strands are in opposite direction.

(b)

Trombone model for DNA replication

DnaA recognizes the Ori site and binds to 9 mer repeats and induces positive supercoils.

This causes melting of DNA in the 13 mer repeat region.

DnaB with the aid of DnaC is loaded on to the DNA.

DnaB is a helicase and acts to unwind the DNA.

Hydrolysis of DnaA/ATP to DnaA/ADP causes it to be released from the Ori.

DNA unwinding by helicase generates positive supercoils. DNA gyrase in E. coli relives the topological stress. single strand binding protein binds to single stranded DNA and stabilizes it.

DnaG (Primase) interacts with DnaB and synthesizes a primer.

DNA polymerase III extend sthe 3'-OH end o the primer.

Leading strand synthesis takes place in 3' to 5' direction.

Lagging strand synthesis starts a bit alter as some of the DNA has to be exposed.

looping out of lagging strand DNA occurs.

Each time the DNA is exposed a primer is syntheiszed by DnaG.

DNA plymerase extends the 3' end of primers but stops as it encounters the 5' end of primer ahead of it.

After completion of DNA synthesis primers are removed by RNaseH.

DNA polymerase III fills the gap.

DNA Ligase seals the nick