DNA Damage, Repair and Recombination

Question 1

How can DNA be damaged during replication?

Answer 1

Mutations - UV radiation, ionising radiation and chemical agents (many mutagens are carinogens)

All mutations are random

Question 2

What is in place to avoid DNA damage?

Answer 2

DNA polymerases have proofreading functions and 3’ - 5’ exonuclease activity to remove a incorrect nucleotide
Telomerase - prevents shortening of the chromosome

Question 3

How can UV radiation affect DNA?

Answer 3

It promotes formation of a cyclobutyl ring between adjacent thymine residues = intrastrand thymine dimer
Thymine-cytosine dimers can also form but less frequently

This distorts DNA’s base pairs, interfering with transcription and replication

Question 4

How can ionising radiation affect DNA?

Answer 4

Via direct action on DNA
OR
Indirectly by inducing formation of free radicals - mainly .OH
This leads to strand breakage

Question 5

What are the types of mutations induced by chemical mutagens?

Answer 5

Point mutations (substitution)

a. Transitions - purine (or pyrymidine) is replaced by another
b. Transversions - purine is replaced by pyrimidine or vice versa

Insertion/deletion mutations
These are collectively known as indels

Question 6

How do point mutations occur?

Answer 6

Altered Bases e.g. nitrous acid HNO2, this oxidatively deaminates aromatic primary amines

Bases can be oxidised by many radical by-products of oxidative metabolism e.g. O2- (from H2O2)

Question 7

How do insertion/deletion mutations occur?

Answer 7

Generated by intercalating agents e.g. acridine orange or proflavin
The distance between two consecutive base pairs is roughly doubled by the intercalation
This can therefore result in insertion/deletion of bases

Question 8

What are some types of DNA repair?

Answer 8

Repair by action of a single enzyme
Base excision repair
Nucleotide excision repair
Mismatch repair

Question 9

What are some enzymes that can repair DNA?

Answer 9

DNA photolyases - absorb light via FADH-, to restore pyrimidine dimers to their monomeric form by splitting the dimer using the excited electron from FAHD-

Alkyltransferases - reverses base methylation, by tranferring the methyl group to its own Cys residues

Question 10

What is base excision repair (BER)?

Answer 10

Removal of a damaged base
DNA glycosylases - cleave the glycosidic bond, leaving a deoxyribose residue with no base attached (apurinic or apyrimidinic sites)
The deoxyribose residue is cleaved on one side by AP endonuclease
The gap is filled and sealed by DNA polymerase and DNA ligase

Question 11

What is the most common base excision repair?

Answer 11

Uracil-DNA glycosylase, which excises uracil residues
UDG is very specific as U and T are very similar but DNA bends more readily at uracil e.g. 45° kink when UDG flips it out

UDG remains bound until am AP endonuclease comes along as an AP site is very cytotoxic

Question 12

What is nucleotide excision repair?

Answer 12

It removes a segment of DNA strand
This happens where bases are displaced or have bulky substituents

UvrABC endonuclease (Ecoli) cleaves damaged DNA at 7th and 3rd/4th phosphodiester bonds
This is displaced by UvrD (helicase II) and replaced by Pol I and DNA ligase

Question 13

What conditions are caused by defective NER?

Answer 13

Xeroderma Pigmentosum - the inability of skin cells to repair UV-induced DNA lesions (sensitive to sunlight)

Cockayne syndrome - hypersensitive to UV radiation, stunted growth, neurological dysfunction (neurone demyelination) and appearance of premature aging

Question 14

What is mismatch repair?

Answer 14

Replication mispairings, that evaded DNA polymerases us corrected by mismatch repair

MutS homodimer binds and detects distortion in the DNA helix
If methylated = no errors
Not methylated = errors
MutL homodimer also binds forming MutS2MutL2 complex
The complex recruits MutH activating exonuclease to make a nick on the unmethylated GATC sequence
UvrD helicase and DNA polymerase III holoenzyme then replace the segment

Question 15

What is DNA susceptible to?

Answer 15

Double-strand breaks (DSBs) - generated by ionising radiation or free radicals (by-product of oxidative metabolism)

Question 16

What are the repair mechanisms for DSBs?

Answer 16

Nonhomologous end-joining (NHEJ) - Ku (protein) fits into the major&minor grooves, forming Ku-DNA complexes
The complexes dimerise to align both halves of a DSB, leaving the ends accessible to nucleases, polymerases and ligases

Recombination repair

Question 17

What is another prokaryotic response to UV damage of DNA?

Answer 17

SOS response
Normally LexA (a repressor) is bound at the operator, preventing DNA polymerase transcribing DNA for SOS proteins

But when UV damage activates this pathway…
recA binds to ssDNA, using ATP, becomes activated
Activated recA binds to LexA and this undergoes proteolysis
Removal of LexA, allows RNA polymerase to start to transcribe the SOS operon

This is a last resort as it is error prone

Question 18

What is DNA recombination?

Answer 18

The rearranging or exchange of genetic material between homologous regions - i.e. between chromosomes or between different regions of the same chromosome

Question 19

What is crossing over?

Answer 19

Two homologous chromosomes cross over during prophase 1 of meiosis
This allows exchange of alleles

Chiasmata - a point where the chromosomes have crossed over

Question 20

Describe homologous recombination?

Answer 20

The two aligned homologous DNA duplexes are nicked/cleaved
The nicked strands cross over to pair with the almost complementary strands on the homologous duplex
Once sealed = heterologous DNA
The crossover point is a 4 stranded structure called a Holliday junction

The Holliday junction is solved by either:
Cleavage of strands that didn’t cross over
OR
Cleavage of the strands that crossed over

Question 21

What is RecA’s role in recombination of Ecoli?

Answer 21

RecA binds to DNA forming a right handed helix
Forming this filament with a deep helical groove - exposing DNA bound in its interior

RecA partially unwinds the duplex and exchanges the ssDNA with the corresponding strand on the dsDNA

1. ssDNA binds to RecA - initiation complex
2. dsDNA binds to initiation complex forming a 3 stranded helix intermediate
3. RecA rotates the bases of the aligned homologous strands = strand exchange (using ATP)

Question 22

What is RecBCD’s role in recombination in Ecoli?

Answer 22

RecBCD initiates recombination by making a single strand available

It binds to the end of dsDNA and unwinds it using ATP-helicase function
It then nucleolytically degrades unwound single strands behind it:
3’ end small fragments
5’ end larger fragments
When it gets to the ‘Chi sequence’ every 5kb on the 3’ end - yeilding the 3’ end ssDNA where RecA binds

Question 23

What does RuvABC do in recombination of Ecoli?

Answer 23

RuvABC mediates branch migration from the Holliday junction

RuvB forms a pseudohexameric ring, 30 Å hole, where dsDNA can be threaded through towards the RuvA octamer
The strands are pushed through RuvA, where they rejoin to form vertical dsDNA - before exiting
RuvC then helps the resolution of the Holliday junction - nick the strands
This is sealed by DNA ligase

Question 24

What is recombination used for?

Answer 24

Repairing damaged replication forks and double-stranded breaks in DNA

Question 25

How does recombination repair double stranded breaks in DNA?

Answer 25

The ds break’s, ds ends are resected to produce ss ends
Each of the 3’ ends invades the corresponding sequence of homologous chromosome - forming 2 Holliday junctions
DNA polymerase fills the gaps and ligase seals the joints
Both Holliday junctions are resolved to produce 2 dsDNAs

Question 26

What is the CRISPR-Cas9 system?

Answer 26

CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats
A system for editing and regulating genomes

CRISPR are transcribed into RNA - crRNA - which is bound by Cas proteins
These complexes bind to dsDNA - at a complemenary genomic sequence, next to a protospacer, and cleave the DNA
Here DNA may be inserted by: nonhomologous end-joining or homology directed repair

tracrRNA is also needed to identify the crRNA to Cas9

Question 27

What are tranposons?

Answer 27

Transposons can move genes between unrelated sites - rearraging sements of DNA
They also promote inversions, deletions and rearragements of DNA
Common in both eukaryotes and prokaryotes
This influences phenotypic expression in the short term and evolutionary development in the long term

Each transposon codes for the enzyme that inserts it into the recipient DNA

Question 28

What are the levels of complexitity of transposons in prokaryotes?

Answer 28

1. Insertion sequences (IS elements) - They generally consist of: transposase gene, regulatory gene and short inverted terminal repeats (IR)
2. More complex ones carry genes not involved in the transposition process e.g. antibiotic resistance genes
3. Composite transposons - gene containing central region with 2 identical IS elements either side
   They can transpose any sequence of DNA in their central region

Question 29

Describe the mechanism of replicative transposons?

Answer 29

1. Staggered cleavage (sticky ends), is made in the target DNA
2. Each end of the transposon is ligated at the insertion site - forming a replication fork at each end of the transposon
3. The transposon is replicated, yeilding a cointegrate (fusion of 2 plasmids)
4. The cointegrate is resolved into 2 separate plasmids, each containing the transposon
   (catalysed by resolvase)

Question 30

What is significant about eukaryotic transposons?

Answer 30

They ressemble retroviruses