Lecture 21 - DNA damage and repair Flashcards
What are the 2 types of mutations?
1) Point mutations: substitution of one base pair for another
Purine for purine/pyrimidine for pyrimidine: transition
purine for pyrimidine/pyrimidine for purine: transversion
2) Insertions/deletions
What occurs during deamination?
Adenine gets deaminated, becomes hyoxanthine, hypoxanthine then bonds with cytosine (A-T –> G-C)
Fixed by base excision repair
What does an alkylating agent do?
adds a methyl group to purines
O6-methylguanine adds methyl group to guanine (G-C –> A-T)
Repaired directly by 06-alkylguanine transferase transfers methyl group to cysteine in active site –> permanently deactivates enzyme
How are oxidative hits repaired?
DNA glycosylases using base excision repair
reactive oxygen speciess produced during aerobic metabolism,
what happens during UV damage?
creates thymine dimers between adjacent bases, cleaved by photolyases that use visible light to hydrolyse bonds
Direct repair
In humans, repaired by nucleotide excision –> cleave out nucleotide then nucleotides on either side
What are the 5 types of DNA repair?
1) Direct repair –> damaged bases repaired on the spot (O6-alkylguanine transferase, photolyase for thymine dimers)
2) Base excision repair –> base is removed and replaced (deamination) base removed first, then entire nucleotide
3) Nucleotide excision repair –> nucleotide removed and replaced (thymine dimers in eukaryotes)
4) Mismatch repair –> distinguish between correct template and incorrect newly synthesized strand
5) Post-replication repair –> involves recombination
What is the basic mechanism for repair?
Endonuclease nicks ssDNA near damage, exonuclease excises damaged section, polymerase fills in gap using complementary undamaged strand as template, ligase seals gap
What is genetic recombination used for?
increase genetic diversity through homologous recombination
repair double strand breaks
restart stalled replication forks
generate immune repertoire
integrate viral genomes into host genomes
Describe the Holliday model
Endonuclease nicks one strand on each pair of homologous chromosomes, strand invasion when nicked strands cross over to complementary strand, branch migration. Resolved using resolvase enzyme. Isomerizes (twists) and then can be cut vertically or horizontally. One way leads to recombinant heteroduplexes, other way leads to nonrecombinant heteroduplex (no genes swapped)
What does the RecA recombinase protein do?
Helps pair homologous DNA, form Holliday intermediates, branch migration.
Pairs to ssDNA (3nucleotides/monomer) invades dsDNA complex and spools it such that it unwinds and rewinds around the ssDNA strand.
How does RecBCD work?
fixes ds breaks. Has helicase and exonuclease (in both directions) activity, when it hits Chi sequence, leaves a 3’OH ss end, which then crosses over and invades homologous chromosome to repair both strands (double Holliday junction).
