Gene-L8-DNA replication III

what types of errors occur during DNA replication and what are the consequences? (2)

1. mismatch

• incorporating the wrong base and substitutions can lead to mutations

• protein alterations can alter amino acid residue and can cause change in gene expression

2. slippage

• in repitive DNA usually- where there’s polymerase switching from DNA pol alpha and then to DNA delta or epsilon- misalignment for polymerases

• can cause insertions or deletions

how is high fidelity maintained during DNA replication? 3

1. complementary base pairing- ensures the correct watson-crick

2. proofreading- 3’ to 5’ exonuclease- DNA polymerase checks the last nucleotide. if incorrect, it pauses and removes it

3. post replication mismatch repair- occurs immediately after replication and detects and fixes remaining mismatches

how does complimentary base pairing ensure accuracy during DNA replication? what is it based on? what is the mechanism?

• based on the correct geometry of the Watson-crick base pairs

• correct pairs fit precisely into DNA polymerase activity site- A-G mismatch makes a wrong shape

mechanism

• polymerase active site closes around the correct base pairing

• it uses the amino acid residues to for a hydrogen bond

• if mistached- the active site cannot close properly and there is not proper Hydrogen bonding and the polymerase pauses

• it unwinds DNA about 8bp and strand shifts to 3’ to 5’ exonuclease site and removes the incorrect nucleotide by hydrolysis

• error goes from 10^-3 to 10^-8 error rate

what happens if a mismatch isnt repaired after DNA replication? what happens if a C should have been added instead of a G

• next round of replication can fix the error as a permanent mutation

• outcome depends on which strand is used as a template though

• if new strand- mutation propagates

• if parental stand was used correctly- original sequence is retained

• -              If the old strand is repaired- then mutate persists

  -              If the new strand (with mutation) is repaired- the mutation stops

what is the mutHLS system? where is it present and what does it do? name the 3 components

• mismatch repair system in e.coli

• MutS: scans DNA for mismatches- distortions or bulges and recognises and binds to this error

• mutL: is a linker or mediator and connects must to mutH

• mutH- endonuclease and cleaves the newly synthesised strand

mismatch repair always targets the NEW strand

how does the MutHLS system identify the newly synthesised strand?

• based on the DNA methylation at GATC strand

• adenine is methylated in the parental strand

• after replication the old strand is methylated and the new strand is temporarily unmethylated

• mutH recognises hemimethylated DNA and costs the non methylated strand

• leaves the parental strand intact

what are the steps of mismatch repair in e.coli? 8 steps- 4 sections

recognition:

1. mutS detects the hemimethylated DNA

2. mutL recruits mutH to the nearest GATC site

nick formation

1. mutH nicks the new unmethylated strand

excision

1. helicase unwinds the DNA

2. exonuclease 1 removes the section of new strand with the error

3. SSB proteins stabilise ssDNA

resynthesis and ligation

1. DNA pol III fills in the gap correctly

2. DNA ligase seals the remaining nice

what are the key components of the mismatch repair system in humans?

• recognition by mutS homologous: MSH2, MSH3, MSH6 which form heterdimers and recognises different mismatches

• mutL homologous- MLH/PMS proteins- MLH1/MLH3/PMS1/PMS2- act as mediators and regulators of repairs

• haven’t found mutH homologs yet

how does the human MMR recognise the new strand?

• recognition occurs before replication is fully complete

• relies on nicks in newly synthesised DNA- many nicks in the Okazaki fragments and occasional nicks in leading strand

• repair machinery target the nicked new strands

• likely involves PCNA- sliding clamp protein and increases the adherence of polymerase to DNA

what are defects in MMR associated with?

associated with a deposition to colon cancer

• Lynch syndrome- mutations in MLH1 and MSH2

what causes DNA damage and what are the types? what are they fixed by? 3 types

• sources: internal and external

• internal- oxidative stress, ROS,

• external- UV, x-rays, chemicals and sunlight

types:

1. small base damage from oxidation, xrays- BER- base excision repair

2. bulky lesions- UV damage- NER nucleotide excision repair

3. crosslinks/ds breaks- homologous recombination repair

mechanism of nucleotide excision repair?

recognition

-XPC complex scans DNA for distortions and bulges

unwinding

• recruits TFIIH transcription factor IIH

• has helicase activity and unwinds DNA

stabilisation

• RPA ssDNA binding proteins stabilise the open DNA

• 25bp bubble is formed

• XPG helps stabilise this

excision

• endonuclease XPG and others cut both sides

• remove 24-32nucleotides

• DNA pol fills the gap and DNA ligase seals the nick

how does BER base excision repair work? 5 steps what does it repair?

• repairs small base damage from oxidation or deamination- deamination of 5-methycytosine to thymine GT mismatch

• DNA gkycosylase recognises GT and removes the damaged base by cutting glycosylic bond. leaves an AP site

• AP endonuclease APE1 cuts DNA backbone at 5’ side of AP site

  

• dRP removal- lyase activity with DNA pol beta removes sugar residue

• DNA pol beta fills the gap

• DNA ligase seals the nick

how do chemotherapy agents target DNA and why are cancer ells vulnerable? give an example

• drugs induce DNA damage to kill cancer cells cells- cancer cells divide rapidly and have defective DNA repair pathways which makes them more sensitive to damage

• cisplatin causes DNA crosslinks and nicks- disrupts replication and triggers apoptosis.