DNA Replication Overview

DNA Replication Overview

• DNA replication is the process of doubling DNA before cell division, preserving genetic information encoded in the sequence of bases.
• Semi-conservative replication: Half of the original parent DNA molecule is conserved in each daughter molecule. Each new strand is half "old" and half "new," with antiparallel orientation.
• Replication occurs in three stages in prokaryotes: Initiation, Elongation, and Termination.

Initiation

• Origin of replication (oriC): A fixed point on the chromosome where replication begins.
• OriC structure: A specific DNA sequence (approximately 245 base pairs) with key regions.
  • Four 9-base pair sequences bind to DnaA proteins.
  • Three 13-base pair sequences are rich in adenine-thymine (AT) pairs, causing DNA denaturation.
• Unwinding at oriC: The DNA helix unwinds at this specific site.
• Formation of replication fork: A Y-shaped structure where DNA strands are separated, allowing active synthesis.

Formation of Replication Fork

• The replication process is bidirectional, proceeding from the origin in two directions, creating two replication forks.
• Steps of DNA double helix separation:
  • DNA helicase binds to single strands near the replication fork.
  • It unwinds the double helix, requiring ATP.
• Single-stranded binding proteins (SSBs): Prevent premature rejoining of separated DNA strands.

Primer Synthesis

• Primase (RNA polymerase): Synthesizes short RNA primers complementary to the single-stranded DNA template.
• Primers are needed for DNA polymerase to start DNA synthesis. The substrate for primase enzyme is dNTPs to build the primer.

Elongation

• DNA polymerase III: Initiates the synthesis of the new strand by adding deoxyribonucleotides to the 3' end of the RNA primer.
• Primosome: The complex of primase and helicase binds to single-stranded DNA to allow primase access to the template and makes RNA primers for DNA polymerase III to start replication.
• Leading strand: Synthesized continuously in the 5' → 3' direction, following the replication fork's direction.
• Lagging strand: Synthesized discontinuously in short fragments (Okazaki fragments), away from the replication fork.

Lagging Strand Synthesis

• Synthesis of lagging strand occurs discontinuously in short Okazaki fragments (100-1000 nucleotides).
• DNA polymerase I removes RNA primers and replaces them with DNA.
• DNA ligase seals the nicks in the lagging strand.

Topoisomerases

• Topoisomerases: Enzymes responsible for removing supercoils in the DNA helix, which form ahead of the replication fork, to prevent interference with further unwinding.
• Two types of topoisomerases:
  • Type I: Reversibly cut one strand.
  • Type II: Make transient breaks in both strands to relive DNA supercoiling, requiring ATP.
• DNA gyrase: A specialized type II topoisomerase essential for prokaryotic DNA replication. Target of quinolone.

Termination of Replication

• Replication ends when both replication forks meet at the terminus region (Ter).
• Ter regions contain specific sequences that halt replication forks in one direction.
• A termination protein binds to the Ter region and arrests replication in that direction.

DNA Polymerases

• Requirement for DNA polymerases to function: Template, Primer, 5'-dNTP precursors.
• DNA polymerase III: 5'→3' polymerization activity, proofreading (3'→5') exonuclease activity, removes incorrectly inserted base.
• DNA polymerase I: Removes RNA primers, replacing them with DNA (5'→3' polymerization), proofreading (5'→3') exonuclease activity.
• Different DNA polymerases have different roles during DNA replication.

DNA Replication in Eukaryotes

• Eukaryotic DNA replication originates at multiple sites.
• Multiple origins allow for faster replication.
• Five classes of eukaryotic DNA polymerase: alpha, beta, gamma, delta, epsilon
• alpha polymerase has primase activity initiating DNA synthesis.
• beta polymerase involves in repair mechanisms.
• gamma polymerase replicates mitochondrial DNA and has proofreading activity
• delta and epsilon polymerases synthesizes the leading strand and Okazaki fragments & has proofreading activity
• Histones synthesis occurs simultaneously with DNA replication, forming nucleosomes with the new histones.

Telomeres & Telomerase

• Telomeres: Repetitive sequences at the ends of linear chromosomes.
• Functions:
  • Protect chromosomes from degradation (attack by nucleases).
  • Prevent fusion of chromosomes.
• Telomere shortening: Occurs with each round of cell division.
• Telomerase: Enzyme that adds telomeric repeats to the ends of chromosomes. Prevents shortening of telomeres. Prevents the cell from entering senescence.

Reverse Transcription

• Reverse transcription: The process of transcribing single-stranded RNA into double-stranded DNA.
• Reverse transcriptase: The enzyme that catalyzes reverse transcription.
• Retroviruses: Viruses that utilize reverse transcription for replication.
  • Examples: HIV, hepatitis C virus.

Explore the fascinating process of DNA replication, focusing on its key stages and mechanisms. Understand the concept of semi-conservative replication and the role of initiation points such as oriC. This quiz covers the structure, unwinding, and formation of the replication fork essential for cellular division.