Basic Molecular Genetic Mechanisms 2 (L2) Flashcards
mechanism of DNA replication?
semi-conservative (determined using heavy and light N isotopes?
direction of DNA replication?
bidirectional
function of helicase
unwinds DNA
leading strand
parent strand that runs 3’ to 5’ so that the daughter strand can be synthesized continuously as the fork unwinds
lagging strand
parent strand that runs 5’ to 3’ so that the daughter strand must be synthesized in fragments as the fork unwinds
ligation
when newly synthesized DNA strand portions are linked together
Okazaki fragments
fragments of DNA synthesized on the lagging strand
RNA primase
puts down about 10 bases of RNA onto the DNA to start replication
DNA polymerase alpha
recognizes and binds RNA primer then continues to replicate the next 30 bases of DNA
describe the primer for DNA pol alpha
hybrid of DNA + RNA
why does DNA replication require a primer?
DNA polymerases require a 3’ OH to begin synthesis, so you need RNA in a primer
does RNA require a primer to replicate?
no - already has 3’ OH
PCNA
proliferative cell nuclear antigen - locks polymerase complex on DNA so that the polymerase won’t fall off and so that the polymerase goes in one direction
DNA polymerase delta/epsilon
takes over majority of DNA synthesis after primer is laid down
replication protein A
binds ssDNA to maintain it in extended form - prevents secondary structures from forming before the polymerase has reached that segment
RNAse H
degrades RNA from RNA/DNA primer so that DNA can link together (ligation)
what is the fingers domain of DNA pol responsible for?
mediates polymerization
what is the exonucleanse domain of DNA pol responsible for?
proofreading activity in 3’ to 5’ direction
what happens after the error is removed by exonuclease?
strand goes back to pol domain and synthesis resumes
how much does exonuclease reduce error rate?
100,000 fold
what are the two ways that spontaneous mutation occurs?
deamination and depurination
depurination
purines are less stable inside the cell - they tend to just go away, leaving a sugar + phosphate
deamination
5-methylcytosine -> C4 amine replaced w/ carbonyl -> thymine - then during replication, the new strand is synthesized with an A where there was supposed to be a G (can also happen C deaminated to U)
difference b/w repair of depurination and deamination
none - both repaired by same base excision repair b/c go through same intermediate
three methods of DNA repair
- base excision repair
- mismatch excision repair
- nucleotide excision repair
base excision repair
- DNA glycosylase chops off base from wrong nt - leaves phosphate + sugar
- endonuclease - cuts on either side of sugar + phosphate
- lyase (part of DNA pol beta) - puts in correct nt
- ligase - seals gap, forms phosphodiester bond
difference b/w endonuclease and exonuclease
endonuclease chops from middle
exonuclease chops from end
mismatch excision repair
enzymes remove larger stretch of DNA than just the one wrong nucleotide - then same repair mechanism (DNA pol beta + ligase)
what proteins are often mutated in patients w/ colon cancer and what repair mechanism are they associated with?
MSH2 and MSH6 - part of mismatch excision repair
effect of UV light on DNA
can cross link 2 T’s next to each other, creating a pouch in DNA (thymine dimer)
what is the problem with thymine dimers?
they don’t replicate faithfully - more prone to cancer
nucleotide excision repair
XP proteins recognize thymine dimers, open up dsDNA, cut small stretch that contains error, then DNA pol and ligase come fill the gaps and link the ends, respectively
what causes double strand lesions?
too much ionizing radiation
what are the two repair mechanisms for double strand lesions?
- nonhomologous end joining (NHEJ)
2. homologous recombination repair
NHEJ
- KU proteins recognize broken ends, bring ends together, chop off any protruding nt’s to get a blunt end
- ligase comes in and joints blunt ends together
limitation of NHEJ
can create frameshift mutations if only one or two nt’s are chopped off
collapse of the replication fork is fixed by what mechanism?
homologous recombination repair
basic idea of homologous recombination repair
borrows information from the intact homologous chromosome to fix the broken chromosome - maintains respective position of the nucleotides (faithful)
Holliday structure
crossover made during homologous recombination repair strand invasion - made by ONE phosphodiester bond
what enzyme mediates strand invasion in homologous recombination repair?
Rec A (bacteria) and Rad51 (human)
link b/w homologous recombination repair enzymes and breast cancer?
Rad51 interacts w/ BRCA1 and BRCA2 (tumor suppressor genes) - homologous recombination repair doesn’t fxn properly in patients with breast cancer
steps of homologous recombination repair
- 5’ exonuclease acts on broken end
- intact daughter strand ligated to repaired parental strand in unbroken chromosome
- Rad51-mediated strand invasion
- Branch migration (Holliday structure formation)
- Strands cut at crossover
- Ligate ends
- Rebuild replication fork and continue replication
difference b/w ss and ds homologous recombination repair
ds: 2 Holliday structures and 2 strand invasions
ss: 1 Holliday structure and 1 strand invasion
what is the Holliday structure also used for?
same mechanism for crossing over during meiosis
resolution of Holliday structure in ds homologous recombination repair?
4 possible products - 2 parental, 2 recombinant
difference b/w early and late viral proteins
early: replicate viral DNA
late: produce protein coat - capsid
phage lytic cycle
- adsorption and injection of viral DNA in host cell
- expression of viral early proteins inside cell
- replication of viral DNA and expression of viral late proteins
- assembly
- lysis and release
what does the plaque assay tell you?
each plaque represents cell lysis initiated by one viral particle - estimate how many infectious viruses were in the solution added to the lawn
retrovirus
virus that contains ssRNA that is used to build DNA inside of the host cell (reverse transcriptase)
retroviral life cycle
- fusion of virus w/ host cell
- reverse transcriptase makes DNA from viral ssRNA
- viral DNA transported to nucleus and incorporated into host genome
- transcription and translation - builds retrovirus proteins
- budding off of retroviruses
