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DIRECT DNA Repair Systems

1. Photoreactive repair

   • Repair of UV-induced photoproducts catalyzed by photolyase activated by visible light

   • Found in bacteria, single-celled eukaryotes, plants, and some animals (e.g., Drosophila) but not in humans.

1. Base Excision Repair (BER–N)

   • Removal of an incorrect or damaged DNA base

   • Repair by synthesis of a new strand segment

   • Nick translation:

     • DNA polymerase initiates removal and replacement of nucleotides

     • DNA ligase seals the sugar-phosphate backbone

   1. BP mismatch is recognized by N-glycosylase

   2. Removal of the incorrect BP (i.e. Uracil) and creating of an AP (apyrimidinic) site

   3. AP endonuclease generates a single-stranded nick on the 5' side of the AP site

   4. DNA polymerase removes and replaces several nucleotides of the nicked strand by nick translation

   5. DNA ligase seals the sugar-phosphate backbone

1. Nucleotide Excision Repair (NER) – UV usually

   • Removal of a strand segment containing DNA damage and replacement by new DNA synthesis

     • Usually thymine dimers

   • Often used to repair UV-induced damage to DNA

     • UV repair

   • Enzymes recognize and bind to damaged region

   • Segment of 4-5 nucleotides are removed from damaged strand

     • On the 3' or 5' side of the photoproduct

     • Releases single-stranded 12 nucleotides containing the photoproduct

   • DNA polymerase fills the gap and DNA ligase seals the sugar-phosphate backbone

     • Similar to BER

   • Uses 4 UV repair proteins

   1. UVR AB binds opposite a thymine dimer

   2. UVR B denatures the DNA around the lesion

   3. UVR A leaves ; UVR C binds and catalyzes 3' and 5' cuts

   4. DNA helicase UVR D helps release the damaged single strand ; DNA polymerase and ligase fill and seal the single-stranded gap

1. Mismatch Repair

   • Removal of a DNA base-pair mismatch by excision of a segment of the newly synthesized strand followed by resynthesis of the excised segment

   • Enzymes are sensitive to methylation

   • Studied in E. coli

   • During DNA replication, parental strand is usually methylated while daughter is not

   • Parental = red ;  daughter/new = blue

   1. MutH protein – binds unmethylated daughter

   2. MutS – binds base-pair mismatch and attracts MutL

   3. MutL – connects MutH + MutS

   4. MutH – cleaves unmethylated new daughter strand, generating a single-stranded gap

   5. DNA polymerase synthesizes gap

Damage That Escapes Repair Before Replication

• Mechanisms that can permit replication to progress despite the presence of damage

1. Translesion DNA Synthesis

   • Error-prone repair mechanism

   • Unrepaired DNA damage can block polymerase II

     • Causes it to stall

   • SOS repair:

     • Last resort

       • When DNA is so heavily damaged that DNA polymerase II cannot enter active site

     • Repair system in E. coli used in response to massive DNA damage that blocks DNA polymerase III

     • Activates translesion DNA polymerases V that bypass these lesions and synthesizes short DNA segments

       • Specialized polymerase with NO proofreading abilities = HIGH mutation rate

1. Double-Strand Break Repair (DSB)

   • Double stranded breaks lack a template for DNA repair

     • Both strands are broken, large deletion, no template strand available

   • Can cause chromosome instability, cell death, and cancer

   • 2 mechanisms:

     1. Nonhomologous end joining (NHEJ)

        • Repairs double-strand breaks occurring before replication

        • Error-PRONE

          • Can lead to mutation

        • When DSBs occur, both strands of DNA are trimmed into blunt ends and then rejoined with DNA ligase

          • Trimming leads to a loss of nucleotides

          • Cannot be replaced

            • Produces an intact DNA duplex and allows replication across the repaired region in the upcoming replication cycle, but the repair is often imperfect because resection removes nucleotides that cannot be replaced.

          • May lead to frameshift mutations

        1. X-ray/oxidative damage produces DSB in DNA

        2. Protein complex Ku80 binds DNA ends

        3. Ends are trimmed

        4. DNA ligase ligates blunt ends to reform an intact duplex

1. Synthesis-dependent strand annealing (SDSA)

   • Repairs double-strand breaks occurring after the completion of DNA replication

   • Error-FREE

   • After DNA replication, if one chromatid gets damaged on both DNA strands, the intact sister chromatid can help repair

     • 1 chromatid breaks

     • The other acts like homologous recombination

   • Strand invasion:

     • Strand with the double break can invade the intact sister chromatid

       • Allows intact sister to be used as a template

     • Offers a template to synthesize new DNA

     • Once 1 strand is repaired, it can be used to fix the other

   • Similar process to homologous recombination but repairs DNA

   1. One chromatid undergoes a DSB

   2. A) Nucleases digest a portion of the broken strands.

   1. B) Rad51 binds undamaged chromatid

   2. A) Strand invasion of the sister chromatid creates a Displacement (D) loop

   1. B) a replication fork assembles on the D loop

   2. New strand synthesis occurs using available intact strands as templates

   3. Partial strand excision ; duplexes reform and strands are ligated

NHEJ and SDSA and CRISPR gene editing:

• CRISPR = simple

  • Relies on a cell's natural DNA repair mechanisms to facilitate genetic engineering

  • Inject an embryo with a plasmid or mRNA to express:

    • Cas9 nuclease enzyme

    • Guide RNA to guide Cas9 to genomic target

    • Donor template (synthetic DNA) if a "knock-in" is required

  • Can permanently change germ-line

1. In the absence of a donor template, NHEJ is used

   • Can delete gene of interest = "knock-OUT"

     • Mutates gene of interest by making a cut but not providing a donor template so NHEJ occurs

2. Presence of donor template enables SDSA

   • re-insertion of modified gene = "knock-IN"

     • Transgenic

     • Inject embryo with gene of interest after a cut is made

       • Has homologous ends but a different gene in the middle to replace the break

       • Replicates back in through SDSA

     • HDR changes genomic region by adding new gene

Transposable Genetic Elements (TGE)

• Selfish DNA elements

  • DNA sequences that move within the genome through transposition

    • Facilitated by transposase enzyme

• Like a virus but simpler

• More transposable element activity = more mutation potential

• Different TGEs vary in length, sequence composition, and copy number

• Shared TGE structure:

  • Terminal inverted repeats on its ends (part of TGE)

  • Inserted TGE is bracketed by flanking direct repeats (not part of TGE)

• Categories of transposable elements:

  1. Retrotransposons:         class I

     • Composed of DNA but transpose through an RNA intermediate

     • DNA ➝ RNA ➝ reverse transcribed into DNA

     • Reverse transcribed DNA inserts into a new location

     • Enzyme reverse transcriptase is used

       • Same as used by retroviruses

     • Transpose through an RNA intermediate. Retrotransposons are composed of DNA, but they are transcribed into RNA before transposition, and the RNA transcript is then copied back into DNA by the specialized enzyme reverse transcriptase. The reverse-transcribed DNA is then inserted into a new location, where flanking direct repeats are formed. The reverse transcriptase enzyme copies single-stranded RNA into DNA. Retrotransposons carrying the reverse transcriptase gene can initiate their own transposition, whereas those lacking the gene must utilize reverse transcriptase synthesized by another retrotransposon.

     • Source of increase in genome size in eukaryotes

  2. DNA transposons:         class II

     • Their transposition produces flanking direct repeats at the site of insertion. At a minimum, all DNA transposons carry the transposase gene that produces the transposase enzyme required for the movement of the transposon, but many DNA transposons also carry other genes.

     • Their movement occurs in 2 ways:

       1. Non-replicative transposition: cut & paste

          • Excision of the element from its original location and insertion in a new location

            • Cuts out then jumps

          • Does NOT increase the transposable element copy number or genome size

       2. Replicative transposition: copy & paste

          • Duplication of the element and insertion of the copy in a new location

            • Duplicates then cuts then jumps

          • Increases the transposable element copy number and potentially genome size

1. Staggered cuts cleave the DNA strands of the target sequence

2. Single-stranded ends result from staggered cuts of the target sequence

3. The transposable element is inserted into the target sequence

4. Gaps are filled by DNA polymerase

• Mutagenic effect of transposition:

  • TGEs can generate mutations whenever they insert themselves into crucial genetic regions (coding region, promoter, etc.)

  • Many examples in various forms:

    • Humans – hemophilia A, Coffin-Lowry

    • Plants – round vs wrinkled pea phenotype

    • Animals – drosophila melanogaster

      • TGEs were not found in wild-captured flies until 1960

      • Suggests around 1960 TGEs (P-elements) were introduced into flies and proliferated fast

• P-elements: transposable elements found in Drosophila

  • Utilized in a technique to generate transgenic flies (before CRISPR tech)

  • Process:

    1. Clone gene of interest into plasmid flanked by inverted repeats characteristic of TGE

    2. Inject embryo with plasmid and transposase enzyme

    3. Gene of interest will randomly insert itself into genome of embryo