Week 4 - Issues in DNA Replication

Describe the unwinding problem

The torsional strain and supercoiling that develops when helicase unwinds DNA.

* Is a problem in both circular chromosomes and large linear eukaryotic chromosomes
* Solved by DNA topoisomerase

How does DNA topoisomerase I solve the unwinding problem?

It creates a transient single-strand break (breaks a phosphodiester bond)

Special problem for linear eukaryotic chromosomes

Primase can’t put a primer right on the edge of the lagging strand, so without intervention, genetic code will be lost with each replication.

Telomerase function

Solves replication problem in Eukaryotes using telomeres

Telomeres

Repetitive G-rich sequences (since RNA template has lots of C’s) attached to 3’ end of parental strand template.

Telomeres and Cancer

* Telomeres are abundant in germ cells but not somatic cells
* Loss of telomeres limits the number of rounds of cell division
* most cancer cells produce high levels of **telomerase**

Two mechanisms that control DNA repair

* 3’ → 5’ exonuclease
* strand-directed mismatch repair

exonuclease

3’ → 5’

removes misincorporated nucleotide by causing DNA pol. to pause and move one space 3’ → 5’ (like backspace) to remove incorrect nucleotide

Nucleases

* ribonucleases
* deoxyribonucleases

enzymes that attack nucleic acids

Strand-directed mismatch repair

* When proofreading fails
* initiated by detection of distortion in the geometry of the double helix generated by mismatched base pairs.
* MutS detects it, MutL helps remove a large strand

DNA damage can be caused by

* oxidation
* radiation
* Can cause Pyrimidine dimer (CT, CC, or TT) where two bases bind to each other
* heat
* chemicals
* as well as other cell stressors

Spontaneous Damage

* Depurination
* You can lose a random purine
* Deamination
* randomly losing an amine group and having U instead of C

How mutations arise

After a **deamination** event, when the G is changed to an A the mutation becomes permanent and you can’t go back because U base pairs with an A!

After **depurination**, the A-T nucleotide pair will just be deleted4

Methods of DNA repair

Base excision and nucleotide excision

Base excision repair

* 1 nucleotide problem

Clips off glycosidic bond between base and sugar

AP endonuclease and phosphodiesterase remove sugar phosphate

Then DNA pol. and ligase successfully add new nucleotide to the gap

Nucleotide excision repair

Excision nuclease chops off pyrimidine dimer

DNA helicase, DNA polymerase and DNA Ligase fix bigger gap

DNA Repair of double-stranded breaks

Nonhomologous end joining

* Break repaired with some loss of nucleotides at repair site, since end joining is just done by DNA Ligase

Homologous recombination

* Break repaired with no loss of nucleotides at repair site
* Using undamaged RNA as a template