Biochem LEC 1-3

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fundamental structure of DNA molecule
- 10.5 bp/turn
- 0.34 nm rise/bp
Is most cellular DNA under or overwound?

underwound
affects of underwinding

induces negative supercoiling, decreases LK
affects of overwinding

induces positive supercoiling, increases LK
linking number formula

twist (T) + writhe (W)
Bacterial Type I topoisomerase
- relaxes negative supercoils
- change LK in increments of 1
Bacterial Type II topoisomerases
- change increments of 2
- topo II(makes negative supercoils)
Bacterial DNA gyrase(6)
- type II
- change LK by 2
- ATPase
- generates negative supercoils and relaxes negative supercoils
- has open and closed conformation
- catalyzes a double strand break
Eukaryotic Topo II
- Topo II relaxes both + and - supercoils
- it cannot induce negative supercoils
how is DNA supercoiled

chromatin assembly in negative solenoid supercoil (forms nucleosomes)
Meselson-Stahl experiment

showed DNA was semi-conservative using heavy labeled nitrogen
enzymes involved in DNA replication
- Pol 1- used for DNA repair
- Pol III- used for DNA replication in E.coli
- DNA Ligase- used to repair nicks
common properties of DNA polymerase
- needs a primer with 3' OH
- driving force: H-bonding and base-stacking interactions
- two-metal ion mechanism that supports the α 3 'OH attack of the incoming nucleotide alpha-P
properties of DNA Polymerase I
- low processivity
- catalyzes both polymerization and hydrolysis
- 3' to 5' exonuclease: enhances fidelity
- 5' to 3' exonuclease: functions in RNA primer removal, DNA repair
properties of polymerase III
- primary POL involved in replication in E. coli
- high processivity factor
- consists of 10 different subunits
how are RNA segments synthesized?

primosome
Eukaryotic DNA Replication enzymes
- primase/Polα: function to synthesized RNA-DNA primers for replication (low processivity)
- Pol δ and PCNA: function in lagging strand
- Pol ε and PCNA: function in leading strand
Function/structure of PCNA
- homotrimer and functions like the bacterial III beta subunit
- different amino acid
Mechanism of Reverse transcriptase (RT)
- t-RNA primer base paired to viral genome
- forms a DNA-RNA hybrid
- RNase H breaks apart RNA strand
- DNA Pol adds DNA to double stranded DNA
mechanism of replication initiation in E. coli
- -DnaA forms a right handed (positive) helical filament
- -bends oriC DNA promoting unwinding
- -negative supercoiling in A::T rich repeats(strand separation)
regulation of replication initiation and chromosome paritioning
- Fully methylated oriC binds DnaA and initiates replication
- Hemimethylated oriC- cannot reinitiate
- Dam methylase-methylates new oriC strand and initiation of replication
Function of DnaA
- uses ATP
- binds to the Ori to form a positive(right-hand) supercoil
- results in negative supercoiling in adjacent A::T rich repeats
- promotes strand separation
function of ORC
- origin recognition complex protein in yeast
- atpase
- promotes formation of right-handed DNA helix
- it's bound to ori DNA throughout cell cycle
- hexameric complex
mechanism of cell cycle control in eukaryotic DNA replication initiation
- Cdc6p binds to ORC (early G1)
- MCM2-7 binds to the complex (helicase) and activates replication at G1/S phase
- Cdc6p is phosphorylated and degradated (CDK activity is high)
- ORC and helicase are phosphorylated and replicated

Author

Sheilaj

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Card Set

Biochem LEC 1-3

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Updated

2018-02-12T01:20:41Z

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