DNA REPLICATION

DNA Replication

• During cell division, DNA molecule is duplicated to form two exact copies, which are then passed to daughter cells.
• DNA replication occurs during the synthesis (S) phase of the cell cycle.
• Replication is semiconservative, meaning each daughter double helix contains one parental and one newly synthesized strand.

DNA Structure

• DNA is a double helix with a backbone of sugar-phosphate-base (A, T, G, C).
• A=T and G=C are the base pairing rules.
• The two strands of a DNA molecule are complementary and run in opposite directions.

DNA Polymerase

• DNA polymerase is the central enzyme in DNA replication, discovered in 1956 in E. coli by Körnberg.
• It catalyzes the joining of nucleotides to form the growing DNA chain, requiring a single-stranded DNA template and dNTPs.
• DNA polymerase only synthesizes DNA in the 5' to 3' direction.

Replication Mechanism

• Replication starts from origins of replication (ORC), which are specific nucleotide sequences.
• Identification of origins of replication is recognized by the replication apparatus (initiator proteins).
• In eukaryotic cells, replication origins are spaced at intervals of 30,000 to 300,000 bp and contain specific sequences (A+T rich region).

Unwinding of dsDNA and Formation of Replication Fork

• DNA helicase helps open up the double helix DNA, unwinding the two strands of parental DNA ahead of the replication fork.
• Single-stranded DNA-binding proteins stabilize the unwound DNA strands, serving as templates for new DNA synthesis.
• A replication fork consists of four components: DNA helicase, single-strand binding proteins, DNA polymerase, and primase.

DNA Synthesis

• DNA polymerase initiates daughter strand synthesis, adding nucleotides to the 3' growing tips on each template in opposite directions.
• Primase initiates synthesis of an RNA molecule (primer) that is essential for initiation of DNA synthesis.
• Since DNA polymerase only synthesizes DNA in the 5' to 3' direction, continuous synthesis of two new strands at the replication fork would require one strand to be synthesized in the 5' to 3' direction and the other in the 3' to 5' direction.

Leading and Lagging Strands

• RNA primer on the leading strand only has to trigger the initiation of the strand once.
• On the lagging strand, an enzyme called primase synthesizes numerous RNA primers, each of which triggers the growth of an Okazaki fragment.
• Okazaki fragments are joined together by DNA ligase, creating a continuous strand.

Removal of RNA Primers and Joining of Okazaki Fragments

• DNA polymerase removes RNA primers (with 5'-3' exonuclease activity) and fills the gaps between Okazaki fragments with DNA.
• The resultant DNA fragments are then joined by DNA ligase.

Topoisomerases in DNA Replication

• Topoisomerases prevent DNA tangling by cutting and rejoining DNA strands, allowing them to "pass through" each other.
• There are two types of topoisomerases: Type I produces transient single-strand breaks in DNA, and Type II produces transient double-strand breaks.

Telomere and Telomerase

• The lagging strand is always shorter than its template by at least the length of the primer, due to the "end-replication problem".
• Telomere and telomerase are involved in the replication of chromosome ends.

Eukaryotic DNA Replication

• In eukaryotes, there are many replication origins on the chromosome, and replication begins at some origins earlier in S phase than at others.
• As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules.