DNA Repair I

DNA mutations

replicated (i.e. inherited) change to a DNA sequence, can include

1. a change to the identity of a single nucleotide

2. the insertion or deletion of one or more nucleotides, or

3. the change in the DNA sequence of large chromosomal regions

point mutations

a change to the identity of a single nucleotide

two types: transitions and transversions

in protein-coding DNA, are transcribed into mRNA and translated into protein

transition point mutation

change the base-pair identity from one pyrimidine-purine pair to another pyrimidine-purine pair

for example, A:T to G:C

transversion point mutation

change the base-pair identity from one pyrimidine-purine pair to a different purine-pyrimidine pair

example: A:T to C:G

how do point mutations occur?

DNA polymerase encounters a damaged base on the template strand or it erroneously incorporates and fails to correct a tautomeric form of the dNTP

replication error

point mutation is a result of incorrect incorporation and failure to proof-read a mismatched dNTP during DNA replication

replication error steps

1. a tautomer of the incoming dNTP is incorporated into a daughter DNA strand without engaging proof-reading activity

2. the mismatch evades endogenous DNA repair machinery in the first generation of cell(s)

3. serves as the DNA template being copied to the daughter strand thereby becoming a mutation in the second generation of cell(s)

mutagen induced error

result of parental DNA having a damaged nitrogenous base (i.e. chemical modification) which readily pairs with the point mutation dNTP

mutagen induced error steps

1. parent DNA strand contains an altered nucleotide

2. the altered nucleotide results in a mismatch during DNA replication resulting in the first generation of cell(s)

3. serves as the DNA template being copied to the daughter DNA strand thereby becoming a mutation in the second generation of cell(s)

silent mutation

point mutation to protein-coding DNA does not change the amino acid

missense mutation

point mutation to protein-coding DNA changes the amino acid

nonsense mutation

point mutation to protein-coding DNA yields a stop codon

frame shirt mutation

insertion/deletion (indel) mutations

one or more nucleotides are inserted or deleted to the wild type DNA sequence

results in a change to the reading frame (sequential organization of codons)

how do frame shift mutations occur?

aberrant recombination events or DNA polymerase experiences template slippage

indel mutations resulting from template slippage

thumb domain of DNA pol grasps template and oligonucleotide DNA, however this interaction does not always enforce hybridization between oligonucleotide and template DNA strands

permits secondary structures to form along one of the strands of duplexed DNA

what specifically results in deletions to daughter DNA?

template DNA secondary structures

what specifically results in insertions to daughter DNA?

oligonucleotide DNA secondary structures

what is prone to template slippage?

DNA sequences having a repeating array of codons (triplet repeats)

many examples of human genetic diseases caused by the insertion of triplet sequences, referred to as triplet expansion diseases

triplet expansion genes and disease

these genes/proteins normally have codon repeats, but it is the duplication and expansion of the repeat tract to an abnormal length that dives rise to these diseases, like Huntington disease

what can aberrant chromosome recombination cause?

large-scale mutations, during mitosis or meiosis

chromosome recombination

crossing over

exchange of genetic material between homologous chromosomes, process creates new alleles and increases genetic diversity

recombination events and mutation

can result in deletion of genes or duplication of genes, can also inactivate the expression of genes or produce gene fusions

deletions and duplications

chromosome misalignment (unequal homologous recombination) can result in this of a chromosome region

inversions

chromosome alleles are reoriented in a process referred to as non-allelic homologous recombination

insertions and translocations/substitutions

special recombination processes allow for this of chromosome alleles

alterations to DNA structures are caused by

mutagenic chemicals

enzymes that modify the chemical structure of bases

UV radiation resulting in photochemistry

deamination

spontaneous process involving hydrolysis of cytosine’s 4c-amino group

results in incorrect base-pairing leading to replication error

oxidative damage

to purines and pyrimidines can result in mismatched base-pairing, leading to replication error

photochemistry

pyrimidine (thymine) dimers are cross linked nucleotides that are produced by this involving UV light, resulting dimers can stall DNA polymerase

mismatch repair (MMR)

process replaces mismatched nucleotides that were incorporated during DNA replication by polymerase

can tell parent and daughter strand apart by methylation- when DNA is newly synthesized, the duplex of daughter and parent DNA is temporarily hemimethylated

mismatch repair steps

1. DNA polymerase misincorporates a nucleotide resulting in mismatched, hemimethylated DNA

2. DNA repair enzyme MutS recognizes the structural distortion of mismatched nucoeotides and recruits MutL, forming a complex

3. the MutS-MutL complex scans DNA in both directions for a hemimethylated GATC motif, forming a loop to the site of nucleotide mismatch

4. restriction enzyme MutH is then recruited to cut the unmethylated (newly synthesized) daughter DNA strand

5. UvrD (helicase) unwinds the daughter DNA strand and Pol I exonuclease degrades nucleotides past mismatch

6. DNA pol III replaces nucleotides and ligase joins the nick

direct repair

process corrects pyrimidine dimer damaged by UV light without excising the damaged DNA

process employs DNA photolyase

photolyase

an enzyme with a chromophore cofactor that captures and converts energy from light to perform photochemistry

involved in direct repair of DNA

direct repair steps

1. the chromophore of DNA photolyase is promoted to an excited electronic state through the absorbance of a photon and transfers this energy to the coenzyme FADH

2. in its excited state, coenzyme FADH donates an electron to catalyze the reversal of the cross-linked pyrimidine dimer

3. electron rearrangement completes its redox cycle to regenerate the FADH coenzyme

base excision repair (BER)

common DNA repair process

recognizes damage at the level of a single nucleotide

oxidative damage or alkylated bases can cause this to be used

employs DNA glycosylase

DNA glycosylase

can recognize base mispairing and subtle problems with a single nucleotide (e.g. oxidative damage), including those problems that do not distort the double helix of DNA

base excision repair steps

1. DNA glycosylase recognizes and cleaves a damaged nitrogenous base by hydrolysis of the N-beta-glycosyl bond

2. cleavage of the nitrogenous base leaves an apurinic or apyrimidic site (AP site)

3. the single stranded DNA is then cleaved at the abasic site by AP endonuclease creating a nick with a 3’-hydroxyl group and a 5’-deoxyribose phosphate

4. the nick translation activity of DNA polymerase I is then used to replace the nick with matching nucleotide

5. DNA ligase rejoins the nick

nucleotide excision repair (NER)

common DNA repair process

recognizes larger, bulkier lesions that include pyrimidine dimers and DNA lesions caused by many environmental mutagens that have only recently come into existence

process makes two incision to the damaged DNA strand, a unique enzymatic activity referred to as exinuclease

nucleotide excision repair steps

1. UvrA recognizes the DNA lesion and recruits UvrB to inwind the DNA at the site of the DNA lesion

2. upon dissociation of UvrA, UvrC is recruited to mediate exinuclease activity making 3’ and 5’ nicks on either side of the DNA lesion

3. the helicase II enzyme (UvrD) displaces the section of lesion containing DNA

4. DNA polymerase I replaces excised segment with the matching nucleotides

5. DNA ligase can rejoin both nicks