Repair of Damaged DNA DNA is the only cellular macromolecule that can be repaired DNA damage includes: base modifications nucleotide deletions or insertions cross-linking of DNA strands breakage of phosphodiester backbone Specific repair enzymes scan DNA to detect any alterations Repair protects individual cells and subsequent generations Mechanisms: 1. direct repair - does not require breaking the phosphodiester backbone of DNA 2. Mismatch repair – replaces incorrect bases shortly after replication 3.Base-excision repair- cuts out damaged bases 4.Nucleotide-excision repair – removes larger- scale distortions by excision

Methyl-Directed Mismatch Repair -Most DNA is methylated, particularly at adenosine -Newly synthesized DNA is not methylated for perhaps several minutes -Mismatch repair enzymes recognize incorrect bases close to GATC’s -Cuts them out and replaces

DNA can be damaged by alkylation, methylation, deamination, loss of heterocyclic bases (depurination or depyrimidization) Glycosylases recognize and remove base (leaves an AP site – abasic site) Sugar and phosphate not removed yet AP endonucleases cut backbone Segment is removed and replaced Details are specific to type of lesion and type of AP endonuclease Base-Excision Repair

Hydrolytic deamination of cytosine to uracil Uracil in place of cytosine causes incorporation of an incorrect base during replication DNA glycosylases hydrolyze base-sugar N-glycosidic bonds Deaminated bases are then removed and replaced

Nucleotide excision repair Enzyme complex cuts backbone in two places –Prokaryotes bases apart –Eukaryotes bases apart E. coli uses the ABC excinuclease (catalyzes 2 specific cuts on same strand) Helicase helps remove damaged section Section replaced

Repair after Photodimerization: Direct Repair Double-helical DNA is very sensitive to damage by ultaviolet (UV) light Dimerization of adjacent pyrimidines in a DNA strand is common (e.g. thymines) Replication cannot proceed in the presence of pyrimidine dimers (distort the template strand) Thymine dimers are repaired in all organisms DNA photolyase is primary repair protein

Repair of thymine dimers by DNA photolyase

Homologous Recombination Recombination - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosome Three types 1. Homologous - exchange between sections of DNA with closely related sequences 2. Site-specific 3.Transposition - occurs between unrelated sequences (e.g. Transposons; jumping genes ) Homologous Recombination Three purposes: 1. Recombinational DNA repair 2. DNA organization during meiosis (eukaryotes) 3. Genetic diversity (exchanging alleles) Two Chromatids showing crossover points

The Holliday Model of Recombination

Site-specific Recombination Purposes: 1. gene expression regulation 2. embryonic programmed rearrangement 3. viral & plasmid replication processes Requires: 1. Recombinase enzyme bp recombination site 3. auxiliary proteins Can occur between 2 different DNA strands or 2 points within the same strand 1. Recombinase recognizes recombination sites. 2. Backbone bond are broken. 3. Transient bonds to recombinase are made. 4. Holliday intermediates are formed. 5. Recombination is completed by cleavage and ligation.

Exchange is reciprocal and precise. Recombination sites are preserved. Outcome depend on location and orientation (5'→3') of site 1.On same DNA molecule – inversion or deletion 2.On different DNA’s - recombination 3.On circular DNA’s - Insertion

Tranposition New segment moved to a target site. Either directly moved or a copy is moved (replicative transposition).