DNA Replication: Primase and Replication Bubble

DNA Replication - RNA Primase

• DNA polymerase can only add nucleotides to a growing polymer.
• Primase synthesizes a short RNA chain called a primer.
• The primer is a DNA/RNA hybrid for a short stretch.
• Base pairing rules are followed, but A-U instead of A-T.
• The primer is later removed, replaced by DNA, and the backbone is sealed (ligated).

DNA Replication - The Replication Bubble

• Studying replication is easier by considering one replication fork.
• Synthesis is bidirectional.
• Two replication forks moving in opposite directions give rise to a replication bubble.
• Prokaryotes have one replication bubble, while eukaryotes have several.

DNA Replication - Mechanics of It

• DNA is synthesized by adding nucleotides along one strand.
• The leading strand is synthesized continuously.
• DNA polymerase reads 3' → 5' along the leading strand from the RNA primer.
• Synthesis proceeds 5'→ 3' with respect to the new daughter strand.
• The lagging strand is also synthesized 5'-3' because that is the only way DNA can be assembled.

DNA Replication - Okazaki Fragments

• On the upper template strand, DNA synthesis is continuous.
• On the lower template strand, DNA synthesis is discontinuous, resulting in Okazaki fragments.

DNA Replication - DNA Polymerase

• DNA polymerase can only add nucleotides to a growing polymer.
• It is part of the replisome complex with other proteins, such as PCNA.

DNA Replication - Steps Involved

• Replication begins at the origin of replication.
• The strands of DNA separate to form a replication fork.
• RNA primase produces RNA primers.
• DNA polymerase III extends DNA on the leading strand.
• As the replication fork moves, a new RNA primer is made on the lagging strand.
• DNA polymerase III extends these primers, forming Okazaki fragments.
• DNA polymerase I removes the RNA primer, replacing it with DNA.
• The fragments are joined together by DNA ligase.

DNA Synthesis

• Phosphate diesters are the 'backbone' of DNA and RNA.
• DNA polymerase provides catalysis for the 3' hydroxyl group on the end of the growing DNA strand to attack the α-phosphate of a 5'-deoxynucleoside triphosphate, expelling inorganic pyrophosphate.

DNA Replication - Factors Involved

• Exact error-free replication occurs prior to mitosis.
• Replication takes minutes in prokaryotes and hours in eukaryotes.
• Requirements for replication include a primer, template, enzymes, deoxyribonucleotide triphosphates (dNTPs), and ribonucleotide triphosphates.

DNA Gyrase

• DNA gyrase is a topoisomerase II enzyme.
• It causes a double strand break and loops one strand under another.
• It removes a 'turn' of the DNA.
• DNA gyrase is a critical target for blocking cell replication, particularly with antibiotics and antineoplastics.

DNA Replication - Enzymes and Their Functions

• DNA gyrase (topoisomerase II) relaxes supercoiled DNA.
• DNA helicase breaks the hydrogen bonds between complimentary base pairs.
• Single strand binding proteins maintain the stability of the replication fork.
• RNA polymerase (RNA primase) synthesizes RNA primers.
• DNA polymerase synthesizes DNA.
• DNA ligase seals the backbone of DNA.