25: DNA metabolism Flashcards
what are the fundamental rules of DNA replication
every organism follows these rules: semi-conservative begins at origin and usually proceeds bidirectionally moves 5'-3' semidiscontinuous (lagging strand)
describe semiconservative replication and how we know DNA does it
semi conservative means that one strand is a parental strand and the other is a daughter strand. we know this is the case because of the heavy N and light N experiment where after one replication there’s an intermediate band and after two there is one light and one intermediate band
draw the DNA polymerase reaction mechanism
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3’ OH attacks the alpha phosphate of the incoming nucleotide. causes release of the two phosphates and joining of the new base by a phosphodiester bond.
what is processivity
products never dissociate away from the enzyme. DNA polymerase does not dissociate from the DNA strand after adding a nucleotide
how does DNA polymerase avoid mistakes when replicating DNA?
- accurate Watson-crick base paring results in a shape that is necessary for optimal catalysis by DNA polymerase.
- proofreading exonuclease activity. DNA polymerase has 3’-5’ exonuclease activity in a specific active site that recognizes and removes mispairings
define replisome and types of DNA polymerases
replisome or DNA replicase system: everything, including many other proteins and polymerase, needed to replicate DNA
E coli polymerases:
polA (DNA polymerase I) has clean up and nick translation function. has proofreading 3-5 exonuclease activity and unique 5-3 exonuclease activity (can be manipulated to form Klenow fragment)
polB (DNA polymerase II) has repair function. 3-5 exonuclease activity
polC (DNA polymerase III) is the main replication polymerase, fast! has 3-5 exonuclease activity
how does DNA pol I work
DNA pol I has nick translation function by 5’ - 3’ exonuclease activity. it hydrolyzes a chunk of bases after the nick and replaces the bases then leaves a nick at the end which ligase seals
structure of DNA pol III
big and complex, 17 subunits. Core (2) does the reaction, Beta clamps (3) bind and hold DNA in place, and the rest functions as scaffolding
proteins required for DNA replication
helicase: use energy from ATP to separate strands
topoisomerase: relive stress created by strand separation
DNA binding proteins: stabilize separated strands
primases: synthesize RNA primers
DNA ligases: seal nicks
DNA polymerase: synthesizes new DNA strand
describe initiation of DNA replication
initiation is the only step that is regulated. involves methylation of specific adenine bases within the tandem array of three 13 bp sequences, which is part of the consensus sequence. Another consensus sequence has four 9 bp AT rich sequences for recognition and binding of DnaA proteins. then AT rich regions denature, DNA unwinds, SSB proteins bind, and topoisomerase
describe elongation
leading and lagging strands are synthesized by the same DNA pol III. leading strand continuously made in 5-3 direction while lagging is discontinuously made in 5-3 direction. helicase unwinds. primase lays down RNA primer on lagging strand which is loaded onto new Beta clamp and released. previous Okazaki fragment finishes and releases with its Beta clamp. Then the new Beta clamp attaches to DNA pol II and synthesizes the fragment. DNA pol I removes RNA primers and fills in DNA, leaving nick which is sealed by ligase
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mechanism of DNA ligase
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first ligase is adenylated from AMP or ATP. Then the 5’phosphate in the DNA nick is activated by adenylation. This allows the 3’OH to attacks the 5’phosphate and release the AMP, sealing the backbone
describe termination of replication
involves recognition of specific sequences. Ter sequences are binding sites for Tus proteins. The Ter-Tus complex arrests the replication fork
eukaryotic replication differences
DNA molecules are much larger and complexly organized. most essential features are the same, including many proteins. differences include many sites for replication origin, multiple DNA polymerases used in elongation, and termination involves telomeres
what happens if a protein is damaged? RNA? DNA? does it carry over to the progeny?
protein: degraded by proteasome, does not carry over
RNA: degraded by exosome, does not carry over
DNA: not degraded. will carry over! must be repaired to stay healthy
how does damage arise in DNA? what is the rate?
lesions may be spontaneous (depurination), induced endogenously (reactive oxygen species), induced by radiation, induced by chemicals, or a WC mismatch.
about 1000 lesions occur in 24 hours, with repair mechs it goes down to only 1 of those 1000 becoming mutations (modification that results in changed RNA/protein sequence)
describe the Ames test
Ames test detects DNA mutation potential. Bacteria with a mutation in gene encoding enzyme for His biosynthesis (HISno cells) are plated on media lacking His. Some have spontaneous back mutations that result in the DNA encoding His to work again, these cells survive on the plate. To test: add filter paper with potential mutagen and observe colony growth around it. the greater # of colonies. the greater the number of revertants, the greater the mutation rate
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types of DNA damage and their repair systems
mismatches: mismatch repair
Deamination, alkylation, pyrimidine dimers, oxidized bases: base excision repair
bases with large adducts that result in structural change: nucleotide excision repair
cyclobutane pyrimidine dimers (prokaryotes), suicide methyltransferases: direct repair
breaks in the DNA backbone: recombinational repair and error prone repair
how does mismatch repair work?
- recognition of mismatch (MutS and MutL) within the context of the sequence GATC (may be 100s of bp away)
- movement in both directions fo the MutS/MutL
- MutH cleaves 5’ of lesion only on the non-methylated strand
- removal of nucleotides from nick back to and including the lesion via exonuclease
- fill in with DNA Pol III and ligate. requires ATP
New strand is methylated by Dam methylase after a few minutes and the strands can no longer be distinguished
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how does DNA mismatch repair link to cancer?
mutations in the DNA encoding the repair enzymes causes inability to correct the mismatch. BRCA1 and BRCA2 interact with repair enzymes, control cell cycle, etc. and women who have a defect in them have an 80% chance of developing cancer
describe base excision repair
the damaged base is removed by DNA glycosylase, leaving a hole in the DNA called the AP site. AP endonuclease cleaves the backbone at the site and DNA pol I (which has 5-3 exonuclease activity) removes nucleotides and polymerizes new DNA. DNA ligase seals the nick
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describe nucleotide excision repair
excinuclease cleaves the DNA on both sides of the lesion, helicase unwinds and removes the lesion, then DNA pol I fills in and ligase seals
describe direct repair using photolyase
not present in humans
light excites MTHFpolyGlu which transfers its energy to FADH-. This donates an electron to cyclobutane pyrimidine dimer and causes the dimer bonds to break, leaving monomeric pyrimidines in the repaired DNA. The electron is returned to FADH- to continue the cycle.
slide 10
describe direct repair using methyltransferase
a mutation occurs that adds a methyl to guanine. A specific enzyme for methyl groups is needed to fix. Methyltransferase, with an active thiol group, transfers the methyl from the guanine to the enzyme. The enzyme is only used once and is then destroyed.
3 classes of DNA recombination
homologous genetic recombination: genetic exchanges between 2 DNA molecules that share a region of almost identical sequence. exchange occurs at any sequence (used in repair, segregation of chromosomes, genetic diversity)
site specific recombination: exchange only at a particular sequence
DNA transpositions: short segment of DNA, moves from one location to another
describe homologous genetic recombination role in bacteria and eukaryotes
bacteria: primarily DNA repair, also during mating/conjugation to contribute to genetic diversity
eukaryotes: replication, cell division, and repair. occurs with highest frequency during meiosis. crossing over of chromosomes!
homologous genetic recombination mechanism
a double stranded break in one of two homologs is converted to a larger gap by exonucleases (RecBCD) which degrade 3’ ends less than 5’ due to reaching chi sequence, producing staggered break. An exposed 3’ end pairs (single stranded DNA protected by RecA) with its compliment in the intact homolog (strand invasion) and is extended by DNA polymerase, generating a DNA molecule with two crossovers (Holliday intermediate). cleavage of the Holliday intermediate generates products with recombined DNA
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how does site specific recombination work? enzymes?
recombinase: site specific endonuclease and ligase in one package. similar mech as topoisomerase. it breaks a strand by binding with Tyr residue to phosphate. results in free OH groups on DNA strands which attack the opposite strands. repeat and the end is a recombined DNA strand
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what are transposons
jumping genes that can randomly move and insert into DNA. possible due to terminal repeats on the transposon and transposase which makes staggered cuts in the target site. transposon inserts at the site of the cuts and gaps are filled by replication
what are immunoglobulin genes?
genes that can produce millions of different immunoglobulins which contribute to antibody molecules variable regions. related to transposition. genes have V variable segments, J segments, and C constant segments. the V and J combinations have huge possibility for variation and encode the antibody variable region.
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