DNA Replication in prokaryotes

 ZOOHCC - 501: Molecular Biology (Theory)

Unit 2: DNA Replication

DNA Replication

Prokaryotes have been the subject of extensive research on DNA replication, largely because of their tiny genome sizes and the availability of mutations. A single circular chromosome in E. coli contains 4.6 million base pairs, and all of it replicates in roughly 42 minutes, starting at a single origin of replication and moving in both the circle. This indicates that almost a thousand nucleotides are inserted every second. The procedure moves along rather quickly and without many errors. Proteins and enzymes are used in great quantity during DNA replication, and each one is to the process. The enzyme DNA polymerase, also known as DNA pol, which adds nucleotides that are complementary to the template strand one at a time to the expanding DNA chain, is one of the main participants. Energy is needed to add nucleotides, and it comes from the nucleotides with three phosphates connected to them, much like ATP has three phosphate groups attached. The energy produced when the bond between the phosphates is broken is utilised to create the bond between the new nucleotide and the expanding chain.Three different forms of polymerases are known to exist in prokaryotes: DNA pol I, DNA pol II, and DNA pol III.

How does the replication machinery know where to begin?

It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs.ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi- directionally as replication proceeds. Single-strandbinding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3'-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3'-OH end. Another enzyme, RNA primase, synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand

A replication fork can move 1,000 nucleotides in one second. At the replication fork, DNA polymerase can only stretch in the 5' to 3' direction, which causes a small issue. The DNA double helix is anti-parallel, which means that one of its strands is orientated in the 5' to 3' direction and the other in the 3' to 5' direction. Because the polymerase may add nucleotides in this direction, one strand, which is complementary to the 3' to 5' parental DNA strand, is continually created in the direction of the replication fork. The leading strand is this one that is always being created. Small pieces of the other strand, which is complementary to the 5' to 3' parental DNA, are stretched away from the replication fork.

The lagging strand requires a new primer for each of the brief Okazaki fragments, but the leading strand can be expanded by a single primer. The lagging strand will be oriented generally in a 3' to 5' to 5' to 3' orientation. As the DNA polymerase continues to add nucleotides, a protein known as the sliding clamp keeps the enzyme in place. A ring-shaped protein called the sliding clamp attaches to DNA and maintains the polymerase in place. Topoisomerase works by temporarily nicking the DNA helix and then resealing it to stop the DNA double helix from overwinding as the DNA is opening up to the replication fork. The RNA primers are changed as the synthesis progresses.