12.1 DNA replication mechanisms and enzymes

DNA replication is a crucial process that ensures genetic information is accurately copied and passed on to daughter cells. This section explores the key enzymes involved in replication, including DNA polymerase, helicase, and primase.

The replication process follows a semiconservative model, where each new DNA molecule contains one original and one newly synthesized strand. We'll examine the steps of replication, from initiation at the origin to the synthesis of leading and lagging strands.

DNA Replication Enzymes

Essential Enzymes for DNA Replication

• DNA polymerase catalyzes the synthesis of new DNA strands by adding nucleotides to the growing strand in the 5' to 3' direction
  • DNA polymerase III is the main enzyme responsible for DNA replication in prokaryotes
  • DNA polymerase δ and ε are the primary enzymes for eukaryotic DNA replication
• Helicase unwinds and separates the double-stranded DNA molecule into two single strands, allowing access for other enzymes to initiate replication
  • Helicase uses energy from ATP hydrolysis to break the hydrogen bonds between the complementary base pairs
• Primase synthesizes short RNA primers (8-12 nucleotides long) that provide a starting point for DNA synthesis by DNA polymerase
  • RNA primers are necessary because DNA polymerase cannot initiate DNA synthesis de novo and requires a pre-existing 3'-OH group

Enzymes for Completing DNA Replication

• DNA ligase joins the discontinuous Okazaki fragments on the lagging strand to create a continuous DNA strand
  • DNA ligase catalyzes the formation of a phosphodiester bond between the 3'-OH end of one DNA fragment and the 5'-phosphate end of another
• Topoisomerase relieves the tension and supercoiling that occurs during DNA replication by introducing temporary single-strand or double-strand breaks in the DNA
  • Topoisomerase I introduces a single-strand break, allowing the DNA to unwind, and then reseals the break (prokaryotes and eukaryotes)
  • Topoisomerase II introduces a double-strand break, passes another DNA segment through the break, and then reseals the break (eukaryotes)

DNA Replication Process

Semiconservative Replication and Initiation

• Semiconservative replication is the mechanism by which DNA is replicated, resulting in two identical copies, each containing one original strand and one newly synthesized strand
  • This mechanism was confirmed by the Meselson-Stahl experiment using heavy nitrogen isotopes and density gradient centrifugation
• Origin of replication is the specific site on the DNA molecule where replication begins
  • Prokaryotes have a single origin of replication, while eukaryotes have multiple origins to facilitate faster replication of their larger genomes
• Replication fork is the Y-shaped structure that forms when the double-stranded DNA is unwound and separated by helicase, allowing replication to proceed in both directions

Leading and Lagging Strand Synthesis

• Leading strand is the strand of DNA that is synthesized continuously in the 5' to 3' direction by DNA polymerase, following the movement of the replication fork
  • The leading strand is synthesized in the same direction as the movement of the replication fork, making it a smooth and continuous process
• Lagging strand is the strand of DNA that is synthesized discontinuously in short fragments called Okazaki fragments, in the 5' to 3' direction opposite to the movement of the replication fork
  • The lagging strand is synthesized in the opposite direction of the replication fork movement, requiring the synthesis of short fragments that are later joined by DNA ligase
• Okazaki fragments are the short, discontinuous segments of DNA (1,000-2,000 nucleotides in prokaryotes; 100-200 nucleotides in eukaryotes) synthesized on the lagging strand during DNA replication
  • Okazaki fragments are initiated by RNA primers synthesized by primase and are later joined together by DNA ligase to form a continuous strand