DNA Replication

Leading strand - DNA replication is constant
Lagging strand - DNA replication is made in fragments
Fragment amounts decreased as replication proceeded. These fragments are called Okazaki fragments
The 3' -> 5' direction is synthesized from 5'-> 3' in fragments

DNAP cannot make a new DNA from "nothing", it can only extend from existing DNA or RNA. This existing DNA/RNA fragment is called a primer.

RNA primers contain more errors so its removal is certain, or RNAP needs no primer can explain why RNA primers are used.

DNAP I - contains 5' -> 3' exonuclease and  Klenow fragment (3' -> 5' exonuclease and polymerase)

5' -> 3' exonuclease used to remove RNA primers
3' -> 5' exonuclease used to proofread DNA

Often used to synthesize DNA probe in the lab because of its high accuracy and high polymerase activity.

contains DNA polymerase and 3' -> 5' exonuclease and parts of 5' -> 3' exonuclease

DNA α - Priming of replication of both strands
DNA δ - Elongation of both strands
DNA β - DNA repair
DNA ε - DNA repair
DNA γ - Replication of mitochrondrial DNA

Primase is a RNA polymerase that synthesizes RNA without the conventional promoter
In eukaryotes, DNAP α acts as a primase. One subunit is a primase and another is polymerase.

Eukaryotes use RNA-DNA hybrid as primers for replication. DNAP α makes RNA-DNA hybrid primers by two subunits. DNAP α makes primers for both strands.

Helicase - Binds to replication origin and unwinds dsDNA to creat ssDNA bubble. Works by breaking hydrogen bonds between bases, but this causes supercoiling.

Topoisomerase - releases supercoil by two ways. Topo I cuts ssDNA and lets DNA relax by itself. Topo II cuts dsDNA, much more efficient, usually for chromatin context.

Can occur by replication errors, chemical damages (alkylation) UV damage, radiation, free radicals, etc.

Most errors are fixed by DNAP, but some are not. Mutations are necessary for evolution
DNA's negative charge attracts electrophilic attack, resulting in alkylation.
EMS - a mutagen that changes G/C to T/A
Adds alkyl group on G.

DNA glycosylase can remove a damaged base. AP endonuclease cuts sugar, and DNA phosphodiesterase cuts phosphodiester bond.
New nucleotide is added by DNA polymerase and ligase.

Nicks are made by an excinuclease on both sides of the damaged nucleotide, and a fragment of ssDNA is removed, resynthesized and reconnected by a ligase

UV causes two T residues to be covalently connected.
DNA photolyase will use light energy to break T-T dimers to repair DNA.
Cryptochromes - type of photoreceptors. important for biological clock
DNA photolyases genes were fused to other sequences to create  cryptochromes (CRY)

BMAL1 and clock are bHLH transcription factors that activate PER and CRY genes.
When PER and CRY levels are low, BMAL1 and Clock become more active, but when PER and CRY levels are high, These proteins provide negative feedback to suppress own transcription.
This is essential for sleeping disorders, metabolic disorders, mental diseases and cancer

Origin of replication, where dsDNA is first opened up. Most prokaryotes only have one per circular genome and eukaryotes have multiple Ori.
Ori usually bound by specific Ori-binding proteins regardless of replication status.
Ori needs to recruit additional proteins to start replication

Prokaryotes - DnaA binds to Ori, which brings HU proteins. This complex bends DNA to help melting dsDNA upstream from DnaA box to form the open complex.
Open complex brings in DnaB (helicase) and then brings in DnaG (primase) to start priming. DNAP will then be loaded to form replisome and the SSB binds to DNA to keep the region open. DNAP III holds onto DNA by a β clamp.
The leading and lagging strand are held together by τ dimers. Leading pushes out while lagging  is pulled back

Replisome meets the terminators that are bound by protein Tus. Multiple Tus to prevent overriding.
Problem of circular genome, linked by a cantenane. The circular genomes under melting, repair and decantanation by topoisomerase.
Eukaryotes have ends, but lagging strand cannot finish replication because primers cannot have overhang.

Normal cells can only divide 60 times in culture before becoming senescence. This is caused by the shortening of telomeres

Telomeres - the ends of chromosomes. Repeats of TTAGGG. TERC ( RNA component) and TERT (reverse transcriptase).

TERC binds to G strand overhang of telomere. TERT uses G strand as a primer and TERC as a template to reverse transcribe a new DNA. Telomerase moves forward to make space for the next round of RT. When 3' DNA overhang is long enough, telomerase falls off, primase comes in to make a new RNA primer.
DNAP comes in to synthesize more DNA from RNA primer

Stem cells have abundant telomerases and full length telomeres. Somatic cells have little to no telomerase. One cause of again in cultured cells in the progressive shortening of telomeres.
Telomere shortening eventually activates apoptosis (cell suicide)

90% of tumor cells have active telomerase
dyskeratosis congenita caused by mutation in RNA component in telomerase.
Paradox: Tumor cells often have shorter telomeres, but increased telomerase activity, explained by further mutated tumor cells enjoy longer telomeres and become malignant. Higher telomerase activity targeted by anti cancer, such as telomerase inhibitors, hTERT vaccines. imetelsat shortens telomeres.