14: DNA repair

proofreading

DNApol III has a 3’ to 5’ exonuclease activity

• cutting out the incorrect nucleotide just added

non-bulky damage.

DNA damage including:

• alkylation

• oxidation

• deamination

• depurination

repaired by

• direct repair

• base excision repiar

bulky damage

DNA damage including:

• pyrimidine dimer (e.g., thymine)

• bulky adduct

• base mismatch

• loop

repaired by

• direct repair (pyrimidine dimers)

• nucleotide excision repair (pyrimidine, bulky adduct)

• mismatch repair (base mismatch)

double strand repair

DNA damage including

• double strand break

repaired by

• nonhomologous end joining

• homologous recombination

direct repair

fix non-bulky DNA damages without removing the affected nucleotide or disrupting the DNA backbone

• convert DNA back into correct form

photoreactivation repair steps

1. UV radiation and formation of thymine dimer

2. photoreactivation enzyme (PRE) or photolyase cleaves the cross-linking bond between thymine bases under visible blue light

3. thymine binds back to their complement nucleotide

base excision repair (BER)

eliminates non-bulky damages that don’t distort the double helix and affect individual abnormal bases

• eg. uracil, 3-methyladenine

base excision repair steps

1. during replication a mismatch is recognized by DNA glycosylase

2. DNA glycosylase cleaves the base-sugar bond, freeing the incorrect base

3. AP endonuclease cleaves the phosphodiester bond = a nick (i.e., opening in the double strand)

4. DNA polymerase adds the correct base

5. DNA ligase links the new nucleotide/base by forming phosphodiester bonds = restores double strand integrity

nucleotide excision repair (NER)

mechanism that repairs bulky damages in DNA that distort the double helix

• thymine dimers

• chemically modified bases

• missing bases

• crosslinks

several nucleotides in the damaged strand are removed from DNA and the intact strand is used as a template

nucleotide excision repair steps

1. ERCC4 endonuclease excises the lesion (larger than lesion itself)

2. DNA polymerase synthesizes the missing nucleotides to fill the gap

3. DNA ligase links the newly synthesized fragment by forming phosphodiester bonds

mismatch repair (MMR)

repair system that repairs the newly synthesized strand, not the parental strand

• fixes base-pair mismatch

• doesn’t fix abnormal nucleotide

mismatch repair steps

1. in DNA replication, a mismatched base was added to the new strand

2. methylation of adenine in 5’-GATC-3’ on the old strand

3. mismatch is recognized by protein MutS

4. a complex with MutS, MutL and MutH forms a bridge so the mismatch is brought close to 5’-GATC-3’ sequence

5. an exonuclease (nonspecific) removes nucleotides on the new strand between the GATC sequence and mismatch (within the bridge)

6. DNA pol replaces with normal daughter strand using parental strand as a template

non-homologous end joining (NHEJ)

fixes double strand breaks by simply attaching the two broken strands back together

• disrupts gene of interest

• indels

occurs in any phase of the cell cycle

non-homologous end joining steps

1. DSB = overhang/sticky regions

2. DSB recognized by end-binding proteins and kept close together by protein cross bridge

3. additional proteins digest one of the two DNA strands = deletion of nucleotides possible

4. DNA pol fills gaps

5. DNA ends are ligated by DNA ligase

homologous recombination repair (HRR)

fixes double strand breaks using sister chromatid

• used as a template

• only available during the S and G2 phases of the cell cycle

homologous recombination repair steps

1. DSB

2. needs a non-damaged DNA fragment that acts as a template

3. endprocessing: digestion of short segments of both DNA strands at the break site

4. invasion and exchange of strands between the unbroken (homologous template) and broken sister chromatids

5. the unbroken strands used as template to synthesize DNA

6. the criss-crossed strands (forming Holliday junction) are resolved