Define Semi-conservative, conservative and dispersive
Semi: each parent strand serves as a template for a new strand and the new and old combine to make the new strand
conservative: the double helix services as a template for a new double helix but doesn't contribute
Dispersive: fragments of the original DNA molecule serve as a template for assembling two new molecules each containing old and new parts.
The Meselson-Stahl Experiment
-to determine which hypothesis of DNA replication was correct
-found that DNA replication was semiconservative- each newly made DNA molecule comprises one old and one new strand If dispersive, same heaviness. If semiconservative, First HL, then LL in later generations, if conservative then always LL.
Where does replication start and how does it continue?
Always happens in 5' -> 3' direction, and starts replication bubble from origin of replication and adds DNA outwards from bubble
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
DNA replication uses RNA primers. Why?
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
Eukaryotic DNA Polymerases
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.
DNA helicase and DNA topoisomerase
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.
DNA damage, mutation,
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.
Base excision repair
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.
Nucleotide excision repair
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 damages DNA, and photolyases, and the biological clock
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)
Core components of the human biological clock Clock, BMAL1 and PER, 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
Replication initiation and elongation
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 and telomerase
Telomeres - the ends of chromosomes. Repeats of TTAGGG. TERC ( RNA component) and TERT (reverse transcriptase).
Replication of telomeres
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
Telomeres and aging
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)
Telomeres and cancer
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.
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