6: DNA Replication & Repair

Whats a DNA template strand

makes complementary strand of DNA via base pairing

Whats a replication machine

cluster of proteins that do DNA replication

Whats a semiconservative model

the daughter DNA double helix contains one strand conserved from the parental molecule and one newly synthesized strand

What is the replication origin? how long is it?

where DNA replication begins

100 nucleotide pairs long

Compare bacteria vs human replication origin?

Bacteria: only has 1 single circular chromosome

Human: 10,000 origins (220 per chromosome)

Why do eukaryotes have a lot more replication origin than bacteria?

• Allows rep to take place in atimely manner

  • If we only had 1 it wld take a long time to rep chromosomes

What are initiator proteins

binds to DNA sequences at the replication origin

• opens the 2 DNA strands apart, breaking the hydrogen bonds

• gets proteins that carry out replication

What is the replication fork? looks like? and direction?

• Y shaped where dsDNA has unwound into ssDNA

• 2 forks formed at replication origin

• bidirectional - move away from e/o in opposite directions

Replication bubble?

replication origin center of forks

What is the replication machine?

moves along the DNA at the fork unzipping the double helix and using the DNA strand as a template to make a new daughter strand

How many nucleotide pairs/sec does the replication machine work for bacteria and humans

bacteria = 1000 nucleotide pairs/sec

humans = 100 nucleotides/sec

Why is the replication slower in eukaryotes than bacteria?

Our DNA is much more complex, bulky, harder to move around DNA strand as prokaryotes don’t have to work around histones

• They can js follow a strand instead of making a loop within a loop

What is the DNA pol III

adds nucleotide to the 3’ end of growing DNA strand using complementary base pairing

• builds from 5’ to 3’

How does DNA pol III work?

catalyzes the formation of phosphodiester bonds between the 3’ end of growing DNA strand and 5’ end of the incoming nucleotide

then nucleotides enter as deoxyribonucleoside triphosphate

Whats a deoxyribonucleoside triphosphate composed of? what its role and how

DNA nucleotide + deoxy sugar + nitrogenous base + 3 phosphates

1. provides its own E, w/out the addition of ATP to the growing strand

• hydrolysis releases pyrophosphate by breaking 3 phosphates into pyrophosphate, which breaks phosphoanhydride bond and the E released is the E required for DNA pol III to add on the growing strand

2. monitors the base pairing between nucleotide triphosphates and the template strand

   • to check if the match is correct for DNA pol to go through the conformational change that allows the catalysis of the phosphodiester bond

diff between ATP and deoxyribonucleoside triphosphate

its a deoxyribose rather than ribose suagr

How many errors does DNA pol III do?

one error in every 10^7 nucleotide pairs

What is proofreading

DNA pol correctors errors it makes

• before addiing the next nucleotide, it double check its work

• SEARCH UP POLY AND PROOF READING ARE DONE BY DIFF DOMAINS OF THE ENZYME

What is the diff between leading strand and lagging strand

LEAD:

synthesized continuously in a 5’ → 3’ direction

• moves towards the rep fork

• can be made all the way to the end

LAG:

synthesized discontinously in small pieces

• moves away from the rep fork

• forms OKAZAKI FRAGS

• delayed process

• CANNOT be synthesized till the end

How come the lagging strand can be synthesized all the way to the end?

• primer at the end of the strand is removed, so there is nothing to add DNA nucleotides to

• laggingin strand becomes shorter with each round of replication

• chromosome will become shorter and eventually lose valuable genetic info

What are RNA primers?

• DNA pol only adds nucleotides to an existing strand of nucleotides

Whats a primase? describe it

makes RNA primer

1. 10 nucleotides long

2. complementary to DNA template so (G-C, A-U)

   1. provides 3’ end as a starting point for DNA pol

Describe the primer for leading and lagging strand

LEAD: 1 primer at the replication origin

LAG: new primer needed for e/c Okazaki fragment

Whats nuclease?

degreades the RNA primer

Whats DNA pol I also called? also what does it do

Repair pol

• replaces the RNA primer w DNA as it uses the adjacent Okazaki fragment as a primer

Whats a DNA ligase

joins the 5’ phosphate end of one DNA fragment to 3’ hydroxyl end

• seals the nick to have a continous DNA

What enzyme works behind the rep machine?

1. DNA pol III

2. Primase or RNA Primer

3. uclease

4. DNA pol I

   1. DNA ligase

What works after or can keep up with the rep machine at the repliaction fork

1. DNA helicase

2. SingleStrand DNA binding proteins

3. DNA topoisomerase

   1. sliding clamp

What is a DNA topoisomerase?

• relieves tension in the double stranded DNA caused by excessive twists when replication fork forms

• makes a ss break in the DNA backbone

• reseals the break once tension has been released

What is a sliding clamp?

keeps DNA pol attached to the template

Whats a clamp loader?

locks a sliding clamp around a new DNA double helix

when does a clamp loader get removed and attached?

each time a new Okazaki fragment is made

What are telomeres? what do they permit?

• long repetitive nucleotide sequences added to the ends of the chromsomes

• allows the cell to distinguish between the natural ends of the chromsomes and the random breaks

What is a telomerase?

• enzyme that carries its own RNA template that it uses to add multiple copies of the same repeptitive DNA sequence to the lagging strand template

• an enzyme that adds nucleotides to telomeres, especially in cancer cells.

What does the telomerase replicate?

ends of eukaryotic chromsomes

Whats the telomere vary by?

varies by cell type and age

Whats a telomere-binding proteins?

protect chromsome ends and maintain telomere length

How come rapidly dividing cells keep telomerase fully active? How come some need to reduce their telomerase activtiy?

cells that line the digestive tract needs constantly replicating

bone marrow cells need to geenrate rbc constantly

• telomerase shrink and eventually disappear to stop cells dividing to control it from uncontrolled proliferation of cells (CANCER)