Script for DNA Replication

Script for DNA Replication

The replication fork

The replication of the E. coli chromosome begins at its single replication origin. Both strands are synthesized

simultaneously by a dimer of DNA polymerase III holoenzymes, which together w 23223w2211x ith the primosome, form an assembly

named the "replisome". Here we will step through the synthetic process at a replication fork.

The polymerase directionality problem

All DNA polymerases synthesize DNA in a 5' to 3' direction, that is, they attach incoming nucleotides to the 3' end of the

growing daughter strand. They require a single-stranded template, nucleoside triphosphates, and that the strand they

are extending form a double helix with the template strand. The tricky part for DNA replication is that although the

replisome must move along the chromosome in a single direction, it must copy the DNA strands that it encounters in

both the 5' to 3' and 3' to 5' directions. Therefore, one strand has to loop around in order to come back through the

second polymerase in the correct direction.

Providing a single-stranded template

In order to provide single-stranded templates for the DNA polymerase to copy, DnaB protein uses the free energy

released by ATP hydrolysis to unwind the DNA helix in advance of the replisome. Because of this helix unwinding

function, DnaB and related proteins are called "helicases". Single-strand binding protein then coats the newly unwound

DNA strands to prevent them from reannealing.

Leading and lagging strands

DNA polymerase III now has single-stranded DNA to copy. Each holoenzyme unit copies a strand of DNA, synthesizing

the new strand in the 5' to 3' direction. Leading strand synthesis is always ahead of lagging strand synthesis because

the lagging strand must loop around to enable the DNA polymerase to synthesize in the 5' to 3' direction. The lagging

strand is synthesized in segments called "Okazaki fragments" which are eventually joined by DNA ligase. The leading

strand is synthesized continuously.

Primosome

When the DNA polymerase III holoenzyme copying the lagging strand encounters the previously synthesized Okazaki

fragment, it releases its bound template strand. The synthesis of the next Okazaki fragment is initiated by the synthesis

of a short segment of RNA that is periodically inserted by a protein assembly known as the "primosome". Note that the

primosome must reverse its direction of travel when it synthesizes the RNA primer. After the additional lagging strand

template is unwound, this RNA primer will find its way to the empty holoenzyme, which then extends the strand to yield

the next Okazaki fragment.

Joining the Okazaki fragments

The lagging strand now contains a series of RNA primers that must be removed. DNA polymerase I uses

nick-translation to replace this RNA with DNA. The DNA strands are then joined by DNA ligase. As the replisome

continues to move along the chromosome, successive Okazaki fragments are synthesized and joined to keep up with

synthesis of the leading strand.

Having completed one Okazaki fragment cycle, we have now returned to where this story started, albeit a bit further along

the DNA.