Double-stranded DNA breaks caused by radiation or chemotherapy can be repaired through two main pathways: homologous recombination and nonhomologous end joining. Nonhomologous end joining is error-prone and involves the loss of nucleotides at the joining point, which can potentially lead to mutations. If a replication fork collapses during DNA replication due to a nick in one strand, the damaged strand is digested and the intact strand is used as a template to fill in the missing information before the replication fork is restarted.

1. DNA Damage and Repair

2. DNA Repair
Double stranded DNA break
Ø X- and γ-radiation (Ionizing radiation) and
Ø Some anticancer drugs cause double-strand breaks
Ø These are particularly severe because incorrect rejoining of double strands of DNA
can lead to gross chromosomal rearrangements that can affect the functioning of
gene
Ø To repair double-strand breaks: two system have evolved
Ø Homologous recombination and
Ø Nonhomologous end joining (NHEJ) (Error-prone, lost of several nucleotides)

3. DNA Repair
Nonhomologous end joining (NHEJ)
Ø Error-prone mechanism, several nucleotides are invariably lost at the point of
repair.
Ø If the two fragments are from the same chromosome, the repair process results in
the loss of several base pairs at the joining point
Ø An example of how repair of DNA damage can leads to mutations !
Ø Due to slow diffusion rate of DNA in the viscous nucleoplasm, the correct ends of
a broken chromosome are generally rejoined together (with loss of base pairs)
Ø Most cases small deletions are not harmful (intronic regions)
Ø Other case leads to translocation, produce chimeric genes
§ The devastating effects of double-strand breaks make these the “most unkindest
cuts of all”

4. DNA Repair
Nonhomologous end joining (NHEJ)
The KU70/80 protein complex

5. DNA Repair
Homologous recombination
Ø These mechanisms involve an exchange of strands between separate DNA
molecules
Ø Damaged DNA sequence is copied from an undamaged copy of the same or a
highly homologous sequence on the homologous chromosome in diploid
organisms or the sister chromosome following DNA replication in haploid and
diploid organisms.
Ø In addition to providing a mechanism for DNA repair, similar recombination
mechanisms generate genetic diversity among the individuals of a species

6. DNA Repair
Repair of a Collapsed Replication Fork
Ø If a break in the phosphodiester backbone of one DNA strand (called a “nick”) is
not properly repaired then replicated portions of the daughter chromosomes
become separated when the replication helicase reaches the nick
Light and dark blue: parent strands
Dark red: Leading strand
Pink : Lagging strand

7. DNA Repair
Repair of a Collapsed Replication Fork
Ø 1st step: exonucleolytic digestion of the strand with its 5′ end at the broken end of
DNA, leaving the strand with a 3′ end
Ø 2nd Step: The lagging nascent strand (pink) base-paired to the unbroken parent
strand (dark blue) is ligated to the unreplicated portion of the parent chromosome
(light blue)

8. DNA Repair
Repair of a Collapsed Replication Fork
Ø 3rd step: Strand invasion
• RecA in bacteria
• The homologous Rad51 in eukaryotes, bind to the single-stranded DNA and
catalyze its hybridization

9. DNA Repair
Repair of a Collapsed Replication Fork
Ø 4th step: Branch migration
• Holliday structure, an intermediate in genetic recombination (Robin Holliday).

10. DNA Repair
Repair of a Collapsed Replication Fork
Ø 5th step: Cleavage of the phosphodiester bonds that cross over from one parent
strand to the other
Ø 6th step: ligation of the 5′ and 3′ ends base-paired to the same parent strands result
in the generation of a structure similar to a replication fork.

11. DNA Repair
Repair of a Collapsed Replication Fork
Ø 7th step: Rebinding of replication fork proteins results in extension of the leading
strand past the point of the original strand break and re-initiation of lagging-strand
synthesis (thus regenerating a replication fork)

12. DNA Repair
Repair of a Collapsed Replication Fork

13. Thank you
See you Next week IA !