BLOCK 3: MBG: (3.2) DNA REPLICATION

Replication Regions

• Late Replication regions are dependent on various factors across different organisms.
• In yeast, specific sequences have been identified for late replication regions.
• The functions of these sequences in other eukaryotes are currently under investigation.

Early vs Late Replication

• DNA replication is not uniform throughout the genome.
• Replication Timing Program is cell type specific and established during G1 phase.
• Early replication regions are associated with active gene transcription.
• Late replication regions are associated with lower gene transcription levels.
• Early replication origins are often located between genes or at distinct locations.
• Late replication origins are distributed more randomly.

Initiation of Replication

• There are two stages of replication initiation in yeast: Origin Licensing and Origin Firing.
• Origin Licensing is a regulatory process that controls the location of replication origins.
• Origin Firing is the activation of origins for replication.
• The Pre-Replication Complex (Pre-RC) plays a crucial role in replication licensing.
• Pre-RC contains the Origin Recognition Complex (ORC), which promotes the binding of Cdc6, Cdt1, and MCM helicase.
• MCM helicase binding initiates replication licensing.
• The Pre-Initiation Complex (Pre-IC) is formed after origin licensing.
• Transcription influences DNA replication and can contribute to genomic instability.

DNA Synthesis

• DNA is composed of phosphodiester bonds.
• The 3' hydroxyl group is essential for DNA synthesis.
• Cleavage of the 3' hydroxyl group prevents strand elongation.
• DNA synthesis occurs in a 5' to 3' direction.
• Leading strand synthesis occurs continuously in the same direction as helicase movement.
• Lagging strand synthesis is fragmented due to polymerase moving against the helicase direction and results in Okazaki fragments.

Rolling Circle Replication

• In circular dsDNA, one strand is cut by a nuclease, creating a 3'OH group.
• DNA polymerase extends the 3'OH group.
• The newly synthesized strand displaces the original template strand.
• The displaced strand acts as a template for the complementary DNA strand.

Single-Stranded DNA Virus Replication

• Circular ssDNA viruses utilize rolling circle replication.
• Linear ssDNA viruses use rolling hairpin replication.
• Initial dsDNA circularization is achieved by host polymerases.

DNA Polymerases

• Prokaryotes have five different DNA polymerases.
• DNA polymerase I removes the RNA template during lagging strand synthesis and elongates the strand afterwards.
• DNA polymerase III is the primary elongation enzyme.
• DNA polymerase III is a complex enzyme with multiple subunits (alpha, epsilon, theta, beta, tau, gamma, chi, psi).
• Eukaryotes have 15 different DNA polymerases.
• Pol alpha is a polymerase-primase that synthesizes RNA primers.
• Pol delta is the main polymerase for lagging strand synthesis and primer removal.
• Pol epsilon is the main polymerase for leading strand synthesis.
• Pol beta and other polymerases play roles in DNA repair.

Topoisomerases

• Topoisomerases are enzymes that unwind DNA to facilitate strand separation.
• Type I topoisomerases cut one strand, rotate it around the other strand, and seal the break.
• Type II topoisomerases cut both strands of DNA.

The End-Replication Problem and Telomeres

• Linear chromosomes cannot be fully replicated.
• Limitations of DNA polymerases and the requirement for RNA primers lead to the End Replication Problem.
• Telomeres are repetitive DNA sequences located at the ends of chromosomes that protect against genetic loss during replication.

Primer Removal: Pol Delta and Flap Endonuclease

• RNA primers must be removed during DNA replication.
• Pol delta displaces the RNA primer, forming a flap structure.
• DNA2 processes long flaps into shorter flaps.
• Flap Endonuclease (FEN1) cleaves the shorter flaps.
• DNA ligase seals the gaps in the DNA backbone.

Bidirectional DNA Replication

• DNA is synthesized in both directions simultaneously from a single origin of replication.
• Polymerases moving in opposite directions are bound to an imaginary line that actually represents the helicase, opening the DNA in one direction.
• The polymerase on the leading strand moves in the same direction as the helicase, continuously adding nucleotides to the 3' end.
• The lagging strand polymerase moves in the opposite direction of the helicase, synthesizing short Okazaki fragments that are later joined together.

DNA and Proteins

• DNA influences protein synthesis by providing genetic instructions for building them.
• The cell relies on DNA for essential functions, such as synthesizing the necessary materials for tasks.
• DNA's organization is dependent on proteins like histones and the replication and transcription machineries.

Genetic Information

• The best genetic material can copy and change to adapt to environmental needs.
• Viruses often rely on host cells for their replication machinery.
• Polymerases have specific functions, such as exonuclease activity for removing RNA primers.

DNA Replication in Prokaryotes vs. Eukaryotes

• Prokaryotes use DNA polymerase III for replicating DNA.
• DNA replication in both prokaryotes and eukaryotes occurs bidirectionally, with leading and lagging strands.
• Eukaryotes have more complex polymerases than prokaryotes but still use the same basic mechanism.

Primers and Origins of Replication

• Primers are short RNA sequences that are used to initiate DNA synthesis.
• They provide a 3’ hydroxyl group required for polymerase binding.
• Origins of replication are organized differently depending on the gene's importance.
• Important genes have origins outside their reading frames to ensure continuous replication.

Licensing and Firing

• The origin recognition complex (ORC) identifies all origins of replication simultaneously.
• Licensing involves binding of ORC, helicase, CDT1, and CDC6 to the origin.
• Firing is the initiation of replication at the licensed origins.

Mutations

• Mutations can be beneficial, neutral, or deleterious depending on the gene and function affected.
• Beneficial mutations improve gene function, while deleterious mutations reduce or eliminate function.
• Mutations in somatic cells affect only the individual, while mutations in germ cells can be passed to offspring.

Induced vs. Spontaneous Mutations

• Spontaneous mutations occur due to errors in DNA replication.
• Induced mutations are caused by external factors, such as radiation or chemicals.

DNA Repair

• DNA repair is essential for maintaining the integrity of the genome.
• Replication errors and induced mutations cause damage to DNA, which needs to be repaired.
• DNA repair mechanisms are highly conserved across organisms, reflecting the importance of this process.