DNA double-helix unwinds in the region to be replicated.
Each DNA strand serves as a template for the synthesis of a new strand.
The site where strands unwind is known as the Replication Fork.
Individual deoxyribonucleoside triphosphates (nucleotides) are added sequentially to the growing end of each new strand.
The order of base addition is determined by pairing incoming nucleotide bases to template strand bases.
The outcome is two new semiconservative double strands.
DNA strands must be separated and unwound before replication; the stability of the double helix makes separation unfavorable.
DNA Helicase binds and hydrolyzes ATP molecules, undergoing conformational changes that allow it to unscrew and pry apart the two strands as it progresses along the DNA.
Bind to open single-strand regions of DNA.
Prevent self-pairing or re-annealing while keeping bases accessible for replication.
Synthesizes an initial “primer” strand (10 to 200 nucleotides) onto the template strand.
The primer strand is essential for initiating replication, constructed from rNTPs, resulting in an RNA primer attached to the DNA template.
A new DNA strand is synthesized only in the 5’ to 3’ direction.
For the template strand oriented 3’ to 5’, this results in continuous addition of nucleotides.
This strand is referred to as the Leading Strand.
The complementary template strand is oriented 5’ to 3’, which cannot be synthesized continuously.
Requires synthesis in the 3’ to 5’ direction, which is not feasible.
This strand is synthesized in pieces known as Okazaki Fragments.
Lagging strand synthesis results in short, unconnected sequences.
These fragments must be ligated together by the enzyme DNA Ligase as replication proceeds.
Initial primer synthesis is challenging and prone to errors.
Using RNA allows for easy replacement with DNA, providing an opportunity to correct mistakes.
Initial RNA is assumed to have errors, which are corrected during replacement.
Topoisomerase I: Relieves accumulated strain from supercoiling by making single-strand cuts in the DNA.
Topoisomerase II: Introduces double-strand breaks, allowing one helix to pass through another before resealing the break, thus managing supercoiling during replication.
The replication machinery consists of:
DNA Helicase: Unwinds DNA.
Primase: Synthesizes RNA primers.
Polymerase: Adds new nucleotides to the growing strands.
Single-Strand Binding Proteins: Stabilize unwound DNA strands.
Clamp Loader and Sliding Clamp: Ensure DNA polymerase remains in place during synthesis.
Mutations, or changes in DNA sequences, result from failures in repair mechanisms.
A mutation rate is approximately 1 base per 10^9 nucleotides per replication, and most mutations are silent (not affecting the phenotype).
The rate of replication mistakes significantly exceeds the mutation rate.
Primary mechanisms include:
DNA Polymerase Proofreading: Correct base pairing leads to conformational change and bond formation, while incorrect base pairing leads to stalling and dissociation of the incorrect base.
Exonucleolytic Proofreading: Wrong bases are removed by the nuclease activity of the polymerase when tautomeric forms of bases are bonded, reverting to correct base pairing.
Telomeres prevent DNA molecules from becoming shorter with each replication, avoiding issues at the ends of chromosomes.
Telomerase: A ribonucleoprotein complex that extends the telomeres by utilizing an RNA template for elongation in the 5’ to 3’ direction, allowing DNA polymerase to bind.
Regulated by cells; synthesis of telomerase can be turned off or slowed down with time or defects in chromosomes.
Insufficient telomerase can lead to cell death or replicative senescence, contributing to certain cancers.