
DNA Damage and Repair • Why do we care? • Genetic diseases • Cancer
Cellular Responses to DNA Damage • Reversal of DNA Damage • Enzymatic photoreactivation • Ligation of DNA strands • Repair of photoproduct • Tolerance of DNA Damage • Replicative bypass of template damage with gap formation and recombination (gap repair) • Excision of DNA Damage • Base excision repair • Nucleotide excision repair • Mismatch repair
Mutagens and Carcinogens • Essentially all mutagens are carcinogens • Most carcinogens are mutagens
Somatic vs. germ line mutations • Somatic mutations can lead to cancer • Germ line mutations can lead to birth defects • Most mutations cause neither • Some fall in non-coding DNA • Others are silent
Types of substitutions • Missense • Results in an amino acid substitution • Nonsense • Results in a stop codon (TAG, TAA, TGA) • Same sense • No effect (silent mutation)
Types of mutations • Multisite • Point mutations
Multisite mutations • Cause gross chromosome abnormalities • Involve large regions of DNA • Arise during meiosis
Types of multisite mutations • Inversions: ACBDEF • Duplications ABCDEEF • Deletions: ABCDF • Insertions: ABCDSEF • Substitutions: ATCDEF
Point mutations • Involve only one or a few nucleotides • Arise during DNA replication • Require two errors • An error during DNA replication • Failure to correct that error
Types of point mutations • Substitutions: GATC CATC • Insertion: GATC GGATC • Deletion: GATC GTC • Duplication: GATC GAGATC • Inversion: GATC GTAC
What is the first defense against mutations? • 3’ to 5’ exonuclease activity of the polymerases
Natural causes of mutations • Base tautomerization • UV damage • Spontaneous deamination
Generation of a mutation by the adenine tautomer- About every 1 in 104 bases
Spontaneous deamination • Three of the four bases have exocyclic amino groups • Adenosine produces hypoxanthine • Guanine produces xanthine • Cytosine produces uracil
Answer • The reason cells use thymine in their DNA • Is to allow recognition of uracil formed from cytosine • But what about RNA? • RNA is short lived and in many copies.
Chemical mutagens • Chemicals that accelerate the deamination reaction • Base analogues • Alkylating agents • Intercalation agents
Base analogues • 5-bromouracil • Goes in as T • Can base pair with A but also G to a smaller degree
Intercalation • Flat aromatic compounds • Acridine dyes • Ethidium bromide • Cause frame-shifting
Repair mechanisms • We are exposed to mutagens all the time • you would expect repair mechanisms to exist • A number of different repair mechanisms do exist
Repair Mechanisms • In mismatch repair • Incorrect base is identified • On short section of a newly synthesized DNA • Removed, and replaced • by DNA synthesis directed by the correct template. • In excision repair • bulky lesions in DNA • exposure to UV light • removed by specialized nuclease systems • DNA polymerase fills gap • DNA ligase joins the free ends.
Intro to DNA Mismatch Repair • Mismatch Repair Genes • recognition and repair of certain types of DNA damage or replication errors • Function to help preserve the fidelity of the genome • through successive cycles of cell division
Mismatch repair • Occurs just after replication • Improves accuracy 102 - 103 fold • Must distinguish the parent from the daughter strand
History of MMR • System first discovered in bacteria • Partially homologous system in yeast • Marked homology between yeast and higher order organisms • Human MMR genes first described 1993.
DNA Mismatches • Damage to nucleotides in ds-DNA • Misincorporation of nucleotide • Missed or added nucleotides
Acquired DNA Damage M -C-A- -T-A- -G-T- -G-T- Demethylation
Nucleotide Misincorporation -C-A-G-C-T- -G-T-C-C-A- CT substitution -C-A-G-C-T- -G-T-T-C-A- -C-A-G-C-T- -G-T-C-C-A- -C-A-G-C-T- -G-T-C-C-A- correctly copied
nucleotide added -C-A-G-C-T- -G-T-C C-A- -C-A-G-C-T- -G-T-C-C-A- A correctly copied Added Nucleotides -C-A-G-C-T- -G-T-C-C-A- -C-A-G-C-T- -G-T-C-C-A-
Mismatch Repair Genes • Recognition and repair of mismatches • Other functions • Repair of branched DNA structures • Prevent recombination of divergent sequences • Direct non-MMR proteins in nucleotide excision and other forms of DNA repair • MSH4 & MSH5 involved (with MLH1) in meiotic crossover
Human Mismatch Repair Genes • MLH1 (3p21) • PMS1 (2q31-33) • PMS2 (7p22) • MSH2 (2p16) • MSH3 (5q3) • MSH6 (2p16) (=GT Binding Protein)
Mismatch Repair Function • MMR Proteins combine as heterodimers • Recognise and bind mismatches • ATP consumption • Recruit other proteins • Separate, destroy and resynthesise new DNA strand • Mechanism works for up to 20 base pairs
MSH Protein Complexes • MutS (MSH2-MSH6) • GT mispairs and short (1 base pair) loops/deletions • MutS (MSH2-MSH3) • Larger mispair loops and deletions • Some overlap in function • MSH2 loss is greater cancer risk
MLH Protein Complexes • MutL (MLH1-PMS2) • MutL (MLH1-PMS1) • No established function • Can bind other MMR proteins, MSH heterodimers and replication factors • As for MSH2, overlap means loss of MLH1 confers the greater cancer risk
Other MMR Proteins • DNA ligase • Replication protein A • Replication factor C • Proliferating Cell Nuclear Antigen • Exonucleases • DNA polymerase
Defective Mismatch Repair • Defects in MMR Genes and Function • Microsatellite Instability • Cancer development
Defects in MMR Genes • Control sequences Nonexpression • Premature stop codon Truncated protein • Point mutations Altered sequence • Insertions/Deletions Frameshift effects • Somatic loss of second allele
Microsatellite Instability • Simple nucleotide repeat sequences • Length should be stable at any one locus • Poly-A and poly-CA repeat sequences particularly prone to mismatch errors • Alterations in length are a sign of deficient mismatch repair • Also called RER (Replication ERror)
Microsatellite Instability -C-A-C-A-C-A-C-A- -G-T-G-T-G-T-G-T- shortened repeat -C-A-C-A-C-A- -G-T-G-T-G-T -C-A-C-A-C-A-C-A -G-T-G-T-G-T-G-T -C-A-C-A-C-A- -G-T-G-T G-T- CA skipped G-T heteroduplex results
MI Positive Tumours • 90% of HNPCC colorectal cancers • 20% of sporadic colorectal cancers • 30% of sporadic uterine cancers