General Genetics/DNA Replication

Models of DNA Replication
When DNA is replicated, what happens to the old (parent) strands and the new (daughter) strands? The theory that two double-helices are formed, one with both parent strands and the other with both daughter strands, is the conservative theory of replication. The theory that no strands are fully parental or filial but contain patches of old and new DNA after replication is the dispersive theory of replication. The theory that each double-helix will contain one parent and daughter strand is the semi-conservative theory of replication. In 1958, Meselson and Stahl showed that the semi-conservative theory of replication is generally correct.

Bidirectional Replication
DNA replication begins at an origin point. Origin points are typically rich in adenine and thymine, as the bonds between those nitrogenous bases are easier to split. Once replication begins, it moves in both directions from the origin point, forming a replication fork at each point where the hydrogen bonds between the DNA strands are being split.

It is also important to consider what happens to nucleosomes during replication. As DNA is replicated, new histones must be created to condense the newly-formed strand. The Replication-Coupled (RC) Pathway is concerned with the removal and reassembly of nuceosomes during replication. The molecular chaperones CAF-1 ( c hromatin a ssembly f actor) and ASF-1 ( a nti- s ilencing f unction) function at the replication fork and are recruited by PCNA ( p roliferating c ell n uclear a ntigen). Histone octomers are destabilized by the replisome (see below). ASF-1 transfers histones from ahead of the fork to the newly-synthesized strand, whereas CAF-1 transfers newly-synthesized histones to the new strand.

The Replisome: Enzymes involved in Replication
There are more specific enzymes involved specifically in eukaryotic or prokaryotic replication that will be addressed later. For now, we are considering the general functions of common replisome enzymes.

Helicase
Helicases are motor proteins that use ATP hydrolysis to split (or "melt") the hydrogen bonds between the double helix.

Single-stranded binding proteins (SSBs)
After helicase melts the DNA strands apart, the single-stranded DNA (ssDNA) is covered with SSBs to prevent it from forming secondary structures.

DNA-Dependent DNA Polymerase
DNA polymerase can add nucleotides to a DNA strand given that (1) the strand has already been "started" (i.e. there is already a 3' hydroxyl group ready to undergo nucleophilic attack) and (2) there is a supply of nucleoside triphosphates (NTPs). The 3' hydroxyl group attacks the α phosphate on the NTP, producing free-floating pyrophosphate (a molecule consisting of the β and γ phosphate groups from the NTP). The pyrophosphate then hydrolyzes into two phosphate groups to provide more energy for the enzyme.

DNA Primase
If DNA polymerase cannot start strands but can only continue them, then what starts the strand? The replication of DNA strands actually does not begin with DNA, but with RNA. DNA primase is an RNA polymerase that forms short strands of RNA along the ssDNA that DNA polymerase can extend.

DNA Ligase
DNA ligase can seal gaps by forming a phosphodiester bond between a 3' hydroxyl group and a 5' phosphate group. Ligase has a lysine group that acts as a nucleophile and attacks the α phosphate on an ATP molecule. This produces pyrophosphate and AMP (adenosine monophosphate) bound to the ligase by a transient linkage. The AMP is attached to the 5' phosphate group, regenerating the enzyme. The 3' hydroxyl group to attack the phosphate of the 5' phosphate group, forming a phosphodiester bond between the nucleotides and releasing AMP. This could not have happened in the absence of DNA ligase, because there was not a sufficient leaving group for the phosphodiesterase formation.

Topoisomerase
As helicase continues to unwind the helix at the replication forks, the rest of the DNA becomes supercoiled. Topoisomerase nicks and reseals the sugar-phosphate backbone to relieve the tension.