DNA replication

How is DNA replicated

DNA polymerase catalyzes addition of deoxyribonucleotides to a DNA strand

DNA polymerase 1

Primer removal and DNA repair with 5’ to 3’ exonuclease and 3’ to 5’ exonuclease

DNA polymerase 3

Main replicators polymerase with 3’ to 5’ exonuclease

dNTP binding to DNA polymerase

Binding of the correct incoming dNTP indices a conformational change in DNA polymerase

How does pol 3 catalyze strand elongation reaction during DNA replication

3’ OH of one sugar attacks the alpha phosphate which is removed from the other phosphates it’s attached to

What does dna pol 3 require

dNTP, Mg2+, pre existing strand (template), primer

Which direction does elongation happen in

5’ to 3’

How can dna pol correct mistakes

Using the 3’ to 5’ exonuclease activity which removes incorrect nucleotides from the 3’ end of the growing strand by hydrolysis

How does dna pol correct mistakes when it goes so fast

A mismatch results in a stall. This pause gives additional time for the incorrect region to flop into the exonuclease active site where it’s removed

Origin of replication (OriC)

Region of DNA with special features and is the start site of DNA replication

DnaA

Binds to the OriC and initiates the pre-priming complex

SSB

Single stranded binding proteins that prevents reannealing

Helicase

Ring like hexamers that hydrolyze ATP to unwind dsDNA

Primase (RNA polymerase)

Synthesize short RNA primers complementary to the ssDNA

What direction is the leading and lagging strand synthesized

5’ to 3’

Where does dna pol 3 synthesize the leading and lagging strands

Simultaneously synthesized at the replication fork

A mutation impairs the 5’ to 3’ exonuclease activity of dna pol 1. What effect does this have on dna replication

RNA primers will remain on the lagging strand after dna replication

Leading strand

Made continuously by pol 3 in 5’ yo 3’ direction

Lagging strand

Is looped out, starting from an rna primer and pol 3 adds 1000 nucleotides in the 5’ to 3’ direction. Releases sliding clamp, new loop formed, sliding clamp added, primase adds rna primer, pol 3 makes new Okazaki fragments, pol 1 fills gaps between fragments and removes primer with 5’ to 3’ exonuclease activity, Ligase seals fragments

How does Ligase seal Okazaki fragments

Joins the 3’ hydroxyl group and the 5’ phosphate group of two fragments

Mechanism of Ligase

Initial charging, transfer, phosphodiester bond formation

Initial charging

Catalytic lysine attacks the alpha phosphate of the ATP, generating lysyl-AMP adduct and releasing pyrophosphate

Transfer

Adenylated Ligase transfers AMP from lysine to the 5’ phosphate at the nicked backbone

Phosphodiester bond formation

3’ OH at the nick site attacks adenylated 5’ phosphate making a phosphodiester bond and releasing AMP