Molecular Biology - DNA Replication

Unit 2

Two reactions catalyzed by DNAP III Core Polymerase

5 to 3 polymerase activity and 3 to 5 exonuclease activity

What is the 5 to 3 polymerase activity

Catalyze DNA polymerase, rapid synthesis of DNA on leading and lagging strands

What is 3 to 5 exonuclease activity

Fix mistakes during DNA polymerization

How DNAP Enzyme determine whether or not proper base pairing is occuring

Needs to line up properly for phosphodiester bond to be catalyzed

Chemical reaction for polymerase activity

The Mg ion deprotanate the 3 OH group to create nucleophile(O), the O- attacks phosphorus atom, bond breaks leaving pyrophosphate as byproduct

What is chemical reaction for exonuclease

Hydrolyzes the mispaired base, the 3 terminus repositions back to polymerase site and polymerase incorporates correct nucleotide

Importance of Pyrophosphate

Two inorganic P, irreversible reaction to keep DNA built foward and permenant.

Helicase

Protein made of six individual monomers in a ring around DNA and pulls apart base pairs in template strand of DNA

Single stranded binding protein

Protein binds to single stranded DNA keeping it in single stranded form for DNA polymerase

Primase

Protein that will synthesize short RNA primers used to initiate DNA replication

Beta Clamp

Helps DNA polymerase protein stay associated with the DNA

Steps of DNA Initiation (Prokaryotes)

DnaA protein initiate unwinding of the A-T rich 13 mers, Helicase binds to the unwound DNA, primase binds to helicase and helicase moves along DNA unwinding it, primase syntehsizes a short RNA Primer.

Clamp Loading Process

placing slidign clamp protein onto DNA so DNA polymerase can attach and replicate DNA efficently.

A-T Rich 13 mer repeats

Short sequences rich in A-T base pairs, site of initial strand separation

DnaA 9 mer sites

DnaA initiator protein binds - multiple bndaA molecules bind cooperatively and wrap DNA, start signal for replication

How does datA ensure initiation occurs only once

DnaA bind to Oric, can also bind to datA. Once replication starts, datA is replicated and opens up more binding sites, move to free datA signals, which bind tigther and stuck at datA .

How does seq A ensure initiation occurs only once

Takes Dna and hides it, can’t initiate.

Consequence of DNA replication terminating outside the terminus region

Disrupts controlled fork convergence, leading to incomplete replication

How is it ensured termination of DNA occurs in the terminus region

Ter proteins, replication forks are not allowed to travel past ter sites oriented in the opposite direction

Differences between eukaryotic and prokaryotic DNA

Eukaryotic genome is much bigger and the genome is linear

How does initiation of DNA replication differ in eukaryotes vs prokaryotes

There are multiple origins of replication

How does termination fo DNA replication differ in eukaryotes vs prookaryotes

Prokaryotes have terminus region, eukaryotes have end replication problem

Telomerase

Extends ends of chromosomes

POT 1 role

controls the length of telomerase, fi too much POT1 blocks if become short POT1 decreases

Relation between telomere length and POT1

The more POT1 the longer the telomere

Besides protein what else is included in structure of telomerase

RNA - acts as primer to synthesize new telomer DNA

Relation between T/S ratio and telomere length

longer telomer longer T/S ratio

What does TRF measure telomere by

Shows length via electrophoresis in shell

Draw backs in FISH and TRF

Visual data that needs interpretting vis the T/S ratio