Bacterial replication

During DNA replication, one original (parent) DNA strand is used as a template to make a new complementary strand. Because the parent DNA is double-stranded, replication produces two double-stranded DNA molecules.

Replication begins at the origin of replication. It is bidirectional, meaning replication proceeds in two directions away from the origin, creating two replication forks. At each fork, the two template strands are copied in opposite ways:

• The leading strand is synthesized continuously.
• The lagging strand is synthesized discontinuously.

To summarize, replication involves these main steps:

• The parent DNA double helix uncoils.
• The two strands separate as the hydrogen bonds between complementary bases break.
• Two new strands are synthesized by complementary base pairing.

Unwinding and stabilizing the DNA

DNA helicases unwind the DNA helix, forming a Y-shaped replication fork.

Single-strand binding proteins attach to each separated strand to prevent the strands from rejoining.

As the replication fork moves forward, the DNA ahead of it becomes overwound, creating positive supercoils. Topoisomerases relieve this strain by temporarily breaking the DNA and then rejoining it, which counteracts the positive supercoiling.

Building the new DNA strands (direction matters)

New DNA nucleotides pair with the template strand through hydrogen bonds. The nucleotides are then linked together into a continuous sugar-phosphate backbone by phosphodiester bonds, which are formed by DNA polymerase.

DNA polymerase forms each phosphodiester bond by joining:

• the phosphate group on the 5’ carbon of the incoming nucleotide
• to the OH group on the 3’ carbon of the nucleotide already in the growing chain

Because of this chemistry, DNA can only be synthesized in the 5’ to 3’ direction. That means the template strand is read in the 3’ to 5’ direction.

Leading strand vs lagging strand

At a replication fork:

• The template strand that runs 3’ to 5’ can be copied continuously, so the new strand made on this template is the leading strand.
• The template strand that runs 5’ to 3’ must be copied in short segments, so the new strand made on this template is the lagging strand.

The lagging strand is synthesized as short pieces called Okazaki fragments. DNA ligase then joins these fragments together.

Primers and the roles of polymerases

DNA polymerase can only add nucleotides to an existing strand; it can’t start a new strand from scratch. To begin synthesis, a short starting segment is needed.

An RNA polymerase complex called primase makes this starting segment by building a short RNA primer complementary to the DNA template.

After the primer is in place:

• DNA polymerase III replaces primase and extends the strand by adding DNA nucleotides to the RNA primer.
• DNA polymerase II digests the RNA primer and replaces the RNA nucleotides with DNA nucleotides.
• DNA ligase joins the remaining DNA fragments to make a continuous strand.