Issues in DNA Replication and DNA Repair Mechanisms

3 Issues in DNA Replication

1) What happens when DNA is unwound?

2) What happens at the ends of eukaryotic linear chromosomes during replication?

3) How are mistakes found and corrected?

The unwinding problem

• Why does it happen

• Where is this problem more evident

• What prevent tension? Does it work forever?

• Main solution - what enzyme is involved

• Supercoiling and torsional strain increases during unwinding process

• Even though the DNA wants to unwind to relieve tension during unwinding, the DNA strand is very long and proteins involved that it is hard to unwind

• Issue in circular chromosome and larger linear eukaryotic chromosomes

• Solution can be supercoiling; however, there can only so much you can supercoil

Main solution

• DNA topoisomerase: many types

  • Will cut the DNA, let the DNA unwind, and then reseals it

Issue at the end of linear chromosomes

• which strand is this a major problem; why isn’t there an issue for the other leading strand?

• Why is shortening of the end of the daughter DNA a problem?

• Issue at the lagging strands

  • In the lagging strand

    • 1) Primase is not very good at putting a primer at the very end

    • 2) The RNA primer gets removed, now you have a 5’ end and you can add on to the 5’ end

  • Results: a shorter replicated strand

• No issue for the leading strand

  • Prof notes

    • Look at the slide from 3.. What is the direction of DNA replication

    • The leading strand primer is removed to fill it in you add on to the 3’ end of the lagging strand of the other replication fork

• Problem: loss of sequence information

Solution to the issue regarding the end of the linear chromosome - Telomerase

The repetitive sequence that is added to the 3’ end of the parental strand (i.e. the lagging strand template) is determined by the RNA template to make a DNA complement in telomerase

• RNA template —> DNA com

Telomere replication process

• what does it not require

1) Telomerase has a bound RNA template that is repetitive for the lagging strand template

2) Resembles:

Reverse Transcriptase (reverse transcription)

3) Generates: G-rich ends (RNA template will have a lot of C’s)

4) telomere adds nucleotides to: 3’ ends of parental strand template during DNA synthesis

For the regular process:

• the DNA synthesis for the lagging strand will

• the removal of the primer results in the lose of repetitive DNA sequence from the RNA template of the telomerase during ligase process

Telomeres and Cancer

•Telomerase abundant in stem and germ-line cells, but not in somatic cells

•Loss of telomeres, which occurs normally during DNA replication limits the number of rounds of cell division

•Most cancer cells produce high levels of telomerase

How are mistakes found and corrected?

• With a lot of DNA replicated, there will be a mistake eventually

• Repair

  • If it doesn’t occur, then later on there is no way to detect there is an issue - a permanent DNA mutation

THE HIGH FIDELITY OF DNA REPLICATION

• RNA polymerases have an error rate of 1 in 10^4

• However, DNA polymerases have an error 1 in 10^9

• For the genome, error is 3 nucleotides every time a cell divides

DNA proofreading and repair

• 2 mechanisms - list only no def

1) 3’ to 5’ exonuclease

• It removes the misincorporated nucleotide

• The DNA polymerase have exonuclease activity

• “chop off a nucleotide at the end immediately“

• 3’ to 5’ backwards reading

• “backspace button or 3’ to 5’“

• DNA polymerase has two active sites

  • Polymerizing site

  • Editing/Exonuclease site

Strand-directed mismatch repair in eukaryotes

• what about leading strand with no nicks?

2) Strand-direct mismatch repair

• occurs when the proofreading fails “backspace fails”

• MutS protein recognizes and sticks to the distortion in the geometry of the double helix

• DNA ligase does not seal it, so MutL see the nick and knows its the synthesized strand

• MutL removes the DNA strand

• DNA synthesizes the gap during ligase process

Different between eukaryotes and prokaryotes

• Difference is un-methylated adenines

Leading strand

• Nick is on the other side of the replication fork

DNA Damage

• why does it happen

• What does it cause

• why factor can damage DNA

• Even after synthesis, DNA can get damaged and need repair

• defects in repair mechanisms leads to human diseases

  • e.g. breat, colon, skin cancers

• Factors

  • UV: causes pyrimidine dimers; two pryimidine dimers causes a covalent bonding when beside each other

  • Causes/Factors

    • **finish notes: Oxidation

Spontaneous Damage to DNA can Also occur

• provide example with water and guanine, another example with cytosine

• 2 Types of spontaneous damage

Depurination

• loss of base, only sugar and phosphate remains

• Could be adenine or guanine

• Could be completely removed because they do not know what it is

Deanimation

• Unique specifically for cytosine

• amine lost results in the creation of uracil

How mutations arise with spontaneous damage

• if the deamination does not get fixed, it creates a permanent mutation

DNA repair

• 2 kinds of mechanisms

1) Base excision repair

• Fixes at one nucleotide at a time

2) Nucleotide excision repair

• Fixes a couple at a time

• Perfect for pyrimidine dimers

• Excision nuclease comes in and creates a large cut

• DNA helicase comes in

• DNA ligase

DNA repair of double-stranded breaks

1) Nonhomologous end joining

• quickly seal together and get going

• Quick + dirty

• Prevent the wrong number chromosomes

2) Homologous recombination

• using the information from the pair to correct

• Slow but accurate