BIOS 21317 DNA Replication and Repair (L4, 5)

DNA replication enzymes

DNA polymerase - synthesizes DNA (14 known varieties in eukaryotes, 5 with known roles)
endonuclease - removes a nucleotide (correcting mistakes)
helicases - unwind the DNA helix
DNA liganse - joins Okazaki fragments and other DNA segments

3 phases of replication

initiation, elongation, termination

initiation

identify origin of replication, license and assemble pre-replication complex, helicases, origin of replication needs to be easily melted (low GC content) and no tendency to bind to itself, ssDNA has a binding protein which helps keep it separate (SSB in bacteria, RPA in eukaryotes)

MCM helicase

homohexamer, ring shift moves the DNA, top subunit then jumps down and pulls new DNA through, off seting of two hexamers helps with melting in eukaryotes

bacterial vs. eukaryotic DNA replication

pretty diverged, conserved only overall scheme for two strand replication, sliding clamp, and clamp hold to hold Okazaki fragments in place

bacterial Pol I connected to

Pol I right next to and connected to exonuclease site

2 metal-ion mechanism

need carboxylate amino acids in the active site which bind to two Mg2+ ions, Mg2+ activates the hydroxyl of the primer which attacks the phosphate, one of the phosphates loses an oxygen, and ligation is complete

sliding clamps

keep DNA polymerases on the DNA as they want to fall off after a period of time, sliding clamps are fairly conserved, called PCNA in eukaryotes

replication on the lagging strand

each starts with a primer, once the DNA polymerase reaches the primer of the previous fragment, exonuclease removes the previous primer and replaces it with DNA, ligase then joins the strands

termination

at the ter sequence, polymerase falls off, for eukaryotes, linear DNA means that the last 5’ end will be shortened, telomers at the end of DNA fragments have many repeated sequences to prevent DNA deterioration, tolemerase can add additional copies to the repeat sequence at the 3’ end when the telomerase needs to be extended (making a longer template)

causes of DNA damage

UV, alkylated/altered bases, chemotherapeutics, AP sites, oxidative damage, hydrolytic attack, methylation

when is DNA damage detected?

during replication by polymerase, damage checkpoints in the cell cycle, repair mechanisms, transcriptional responses

base excision repair (BER)

bad bases are recognized, snipped out leaving ribose and phosphate behind, once a replacement arrives, the leftover portion is replaced, then religated, works for small, nonbulky mistakes, human uracil DNA glycosylase is an example of a BER enzyme that removes uracil (methylated C)

human uracil DNA glycosylase (maybe put on sheet)

recognizes a mismatched U because of the increased amount of interactions (can H bond at 3 sites), also sterics and structural changes (for example, T would have a methyl group where U has nothing)

nucleotide excision repair (NER)

repairs bulk adducts (UV damage, smoking), recognizes a structural change in the DNA (usually with roving scanner or a blocked RNA polymerase), removes mistake and regions surrounding it

TT dimer recognition

TT dimer mistake needs to be removed by NER, recognized easily because protein can insert a loop into the helix cause of weak stacking, protein loop doesn’t touch TT, but the TT just makes space

mismatch repair (MMR)

common for repeating bases, often causes a kink in the strand due to a nonbonded nucleotide, also causes wobble pairings, recognition of newer strand by methylation (new DNA has not yet been methylated), does not work on covalent changes

mechanism of MMR in humans

sliding MutS clamp recognizes mismatch, kinks DNA, and sticks F next to the mismatch, recruits MutL, which nicks the DNA and recruits activated helicase and polymerase

single molecule FRET

flourination which allows us to watch things slide

double stranded DNA breaks

caused by meiosis, ionizing radiation, collapsed replication fork, CRISPR, usually fixed by nonhomologous end joining (NHEJ), NHEJ has many ways to join two dsDNA

homologous recombination (HR)

recognition and strand invasion done by RecA (bact) and Rad51 (Euks) which are ATP dependent DNA binding enzymes, have Walker domains for ATP binding and DNA binding domains

DNA replication and cancer

targetting replication or repair mechanisms that are cancerous, meaning errors are already present, can damage them so much that apoptosis will happen instead of repair, since cancer cells are not stopping through replication, they are also not pausing for repair, which makes them more prone to apoptosis

main treatments for cancer

surgery, ionizing radiation (causes DNA breaks and damage), immune therapy

BRCA1/2 mutations

proteins involved in DNA repair, having a mutation in just one is easier to treat than mutations in both, can take preventative cancer drugs if a mutation is known