MolGen-5

DNA acts as a template for its own replication.
Template strands
- S strand:
- 5' -> 3' sequence: C A T T G C C A G T
- 3' -> 5' sequence: G G T C A (Accompanying template S strand)

Nucleotides: Enter the reaction as deoxyribonucleoside triphosphates.
Addition of a deoxyribonucleotide occurs at the 3' end of a polynucleotide chain.
Base-pairing between an incoming deoxyribonucleoside triphosphate and an existing strand (template strand) guides the formation of the new strand.

An incoming deoxynucleoside triphosphate forms a base pair with its partner in the template strand.
It is covalently attached to the free 3' hydroxyl (3' OH) end of the growing DNA strand.
New DNA strands are synthesized in the 5'-to-3' direction.
The energy for this polymerization reaction comes from hydrolysis of a high-energy phosphate bond in the nucleoside triphosphate, releasing pyrophosphate.

The enzyme DNA polymerase catalyzes the reaction.
It guides the incoming nucleoside triphosphate to the template strand and positions it for reaction with the 3'-hydroxyl group of the newly synthesized strand.
DNA replication is semiconservative: each daughter DNA double helix consists of one old strand and one newly synthesized strand.

Replication forks move in opposite directions on a circular DNA molecule in E. coli.
The lagging strand is synthesized in pieces known as Okazaki fragments, which are displayed as:
- Direction of Fork:
- Lagging-strand template of left-hand fork: 3' to 5'
- Leading-strand template of left-hand fork: 5' to 3'

DNA polymerase proofreads its work.
If an incorrect nucleotide is added, the polymerase stops, cleaves it away, and replaces it with the correct one before continuing.
DNA polymerase contains separate sites for synthesis and proofreading.
Incorrect nucleotides cause the new strand to unpair and move to an editing site (E) for removal.

Specifically, the probability of error is:
- Errors per nucleotide added during 5' to 3' polymerization: 1 in 10^5
- 3' to 5' exonucleolytic proofreading: 1 in 10^3
- Strand-directed mismatch repair: 1 in 10^10

DNA Pol I: Exonuclease activity removes RNA primer, replacing it with newly synthesized DNA.
DNA Pol II: Repair function.
DNA Pol III: Main enzyme that adds nucleotides in the 5'-3' direction.
Helicase: Opens DNA helix by breaking hydrogen bonds between nitrogenous bases.
Ligase: Seals gaps between Okazaki fragments.
Primase: Synthesizes RNA primers needed to start replication; can initiate new chains without a base-paired 3' end.
Sliding Clamp: Holds DNA polymerase in place during nucleotide addition.
Topoisomerase: Relieves stress on DNA during unwinding by causing breaks and resealing the DNA.
Single-strand binding proteins (SSB): Prevents rewinding of single-stranded DNA.

RNA primers are synthesized at approximately 200 nucleotide intervals on the lagging strand, each being about 10 nucleotides long.
DNA ligase joins Okazaki fragments using ATP to activate the phosphate of one fragment before bonding it to another.

Helicases are multi-subunit proteins that bind and hydrolyze ATP, moving unidirectionally along a single strand of DNA.

SSB proteins cover the single-stranded DNA, preventing hairpin structures and stabilizing the DNA for replication.

The sliding clamp holds DNA polymerase around the DNA helix, as shown in E. coli with a DNA structure added to indicate its fit.

The parental DNA must rotate at about 50 revolutions per second when the replication fork is moving at 500 nucleotides per second.
If the tension cannot be alleviated due to fixes on DNA ends, it results in supercoiling.

Topoisomerase creates transient breaks to relieve torsional stress allowing for unwinding.
Type I topoisomerases relieve strain without ATP, while type II enzymes hydrolyze ATP and are crucial for fast-dividing cells.

Initiation requires loading two Mcm helicases during the G1 phase.
During S phase, these helicases are activated, allowing for CMG helicase formation (Cdc45, Mcm, GINS).

Replication is stochastic, meaning that it occurs randomly.
Heteroplasmy: Having multiple mitochondrial allelic variants in a cell.
The D Loop maintains mitochondrial origins for the replication of each DNA strand.

Chromosome III of S. cerevisiae has 18 replication origins used at varying frequencies:
- Red origins: <10% usage.
- Green origins: ~90% usage.

Polymerase	Function	Exonuclease Activity
α	Synthesizes RNA primer and initiates DNA synthesis	None
β	Repair DNA	None
γ	Replicate mitochondrial DNA	3' to 5'
δ	Synthesizes the leading strand; fills in gaps	3' to 5'
ε	Repair DNA	3' to 5'

Telomeres are synthesized by telomerase extending the 3' end using an RNA template.
The composition of telomeres is TTGGGG repeated sequences, with synthesis occurring in the 5' to 3' direction.

The t-loop at the end of a mammalian chromosome forms a loop structure essential for protection against degradation.