DNA Recombination and Repair

Holliday Model of Recombination

Nicks are made in one strand of each chromosome, 3’ ends of cut strands join 5’ end of its homologous strand, branch migration forms a heteroduplex region, DNA is other cut and repaired to form two complete recombinant strands

Double Stranded Break model of recombination

most likely mechanism, double stranded break is generated, processing of ends results in 3’ overhangs, recruitment of homologous DNA for repair, strand displacement results in D-loop formation, strand invasion by 3’ single stranded end, extension of 3’ end using homologous DNA as template, formation of double holliday junctions, branch migration, resolution of double holliday junction

RecA

recombinant protein that binds to single stranded DNA to promote invasion of double stranded helix

RuvA/B

recombinant protein that causes branch migration

RuvC

recombinant protein that cleaves at branches and ligates

point mutations

one nucleotide base is changed

transitions

point mutation where purine is replaced by purine or pyrimidine is replaced by pyrimidine

transversions

point mutation where purine replaces a pyrimidine or vice versa

missense mutations

point mutation that causes an amino acid substitution

nonsense mutations

point mutation that causes a premature stop codon

Insertions/Deletions

mutation where large or small segments of DNA are inserted or deleted from the genome

Double stranded breaks

DNA breaks at both strands, body cannot tolerate these so they will be repaired as fast as possible or initiate cell death

Endogenous mutations

mutations that occur naturally in the body, replication errors, errors caused by metabolic by products

Exogenous mutations

mutations that are caused by outside factors; mutagens, carcinogens, ionizing/non-ionizing radiation

Reactive Oxygen Species

superoxide (O2-), hydrogen peroxide, hydroxyl radical; creates 8-oxo-guanine which can replace guanine in DNA

Mutagenesis by nitrous acid

causes deamination; changes cytosine to uracil and adenine to hypoxathine

mutagenesis by ethylmethane sulfonate

alkylation, changes guanine to 6-ethylguanine

Direct Reversal (DR)

DNA repair pathway absent in humans, CPD photolyase binds to a dimer and absorbs light causing a reversal of a pyrimidine dimer and release of the enzyme

Base Excision Repair (BER)

occurs if there is only one damaged base, DNA glycosylase removes damaged base creating AP site, AP endonuclease removes a few bases from around AP site, DNA pol and ligase fill in the gap

Nucleotide Excision Repair

Repairs bulky damage like pyrimidine dimers in humans, major human defense against sunlight and tobacco smoke; endonuclease makes 2 cuts, ~7 upstream and ~3-4 downstream from error region and removes the entire region, UvrD binds to displace this fragment, DNA pol I and ligase fill the gap

Mismatch repair

corrects replications errors of insertions/deletions up to 4 bp; parental strand is methylated at adenine in 5’GATC, mutS recognizes mismatch, mutL binds and forms a loop in DNA, attracts MutH which nicks at 5’ base on unmethylated strand, bases are removed and the gaps are filled by DNA polymerase and ligase, newly synthesized strand is then methylated

Non Homologous End Joining

Double stranded break is recognized by Ku70/Ku80 dimer and DNA protein kinases, DNA-pks and artemis are phosphorylated, DNA ends processed by a complex that includes artemis and the ends are joined directly; very error prone, last resort

Homology directed repair

double stranded break is recognized by MRN complex, ends are ‘resected’ to give single stranded regions, single stranded searches for homologous region in intact duplex, DNA synthesized from invading end, forms holliday junctions, branches migrate and resolvase resolves holliday junction

Xeroderma pigmentosum

extreme sensitivity to UV light, skin cancer rate more than 2000x the normal rate, autosomal recessive, caused by defect in nucleotide excision repair

Cockayne Syndrome

hypersensitive to UV light, stunted growth, neurological dysfunction, caused by defect in nucleotide excision repair and other pathways

Human nonpolyposis colorectal cancer

causes colorectal cancer, defects in Mismatch repair

Heredity Breast Cancer

caused by defects in BRCA 1 and 2, which are part of the non homologous end joining pathway