BB1720 DNA: replication and recombination Flashcards
What is DNA replication?
- copying of DNA
- occurs during mitosis and meiosis, S phase
importance of DNA replication
DNA is constantly being damaged
damage must be repaired
replication must be accurate
how is accuracy achieved?
complementary base pairing
process of dna replication
- two DNA strands are separated
- each strand is used to make a - new copy of the missing strand
- semi conservative
key components of DNA replication
dNTPs
DNA helicase
single stranded DNA binding proteins
DNA polymerase
what are dNTPs?
- building blocks of DNA
- purines -> adenine + guanine
- pyrimidines -> cytosine + uracil + thymine
- A and T
- G and C
- placed into PCR
What is DNA helicase?
- enzyme that unwinds and separates double helix DNA, antiparallel
- allows DNA polymerase to gain access and make a copy
- requires energy
single stranded DNA binding proteins
- binds to single stranded, lagging DNA
- occurs after helicase unwinds the double helix
function of single stranded DNA binding proteins
- stops strands from being repaired or destroyed
- prevent base pairing
- prevent DNA from sticking to itself and block copying
- prevent formation of hairpins
function of hairpins
stops polymerase from working
DNA polymerase
- makes the copy
- adds dNTPs to 3’ end of the DNA molecule
- chain grows from 5’ to 3’
- DNA is antiparallel
- adds 500-1,000 bases per second and helicase unwinds at the same rate
- makes mistakes
how does the antiparallel relate to DNA polymerase and replication?
- one strand faces the wrong way for replication
- the replication fork constantly exposes new bases
how does DNA polymerase use thereplication fork to create DNA?
- bottom strand of the fork has a constant supply of bases to pair
- top strand runs out of DNA - lagging strand
- 3’ 5’ ends come back into helicase. 5’ ends come out of the top. 3’ come out the bottom
- short fragment of DNA produced
why are single stranded binding proteins required for DNA polymerase replication fork mechanism?
prevent base pairing and DNA primase
add RNA as start point for DNA polymerase
conditions for DNA polymerase to make copy of DNA
- cant make copy until it has the template
- needs 3’ OH group to attach nucleoside triphosphate
- needs a primer - insert U instead of T which is later corrected
what happens when DNA polymerase makes mistakes
proof reading
replication stops until error is corrected
if error isnt corrected it’s repaired later
role of DNA topoisomerase
- helps unwinding
- solves DNA winding problem and supercoiling
- makes small single stranded break to allow DNA to spin around at the bond
- creates problem due to break in backbone - repairs DNA phosphodiesterase bone
DNA primase
builds short RNA primer on DNA template
provide 3’ OH group for DNA polymerase to add the nucleoside triphosphate
inserts u instead of t - corrected
DNA ligase
join DNA strands together
repairs DNA single strand nick
What is DNA recombination?
exchange of DNA strands to produce new nucleotide sequence arrangements
what is the purpose of DNA recombination?
maintain genome integrity
generates genetic diversity
how does DNA recombination occur
between regions of similar sequences by breaking and rejoining DNA segments
what happens during DNA recombination
- homologous chromosomes are nicked at identical locations
- strands from one side of nick invades chromosomes and base pairing occurs
- crossing over between two pieces of homologous DNA (4 strands) known as heteroduplex region
- base pair interactions
- occur either vertically or horizontally
what is the heteroduplex region
D E F no longer match to D’ E’ F’
repaired by mismatch repair
formed at the cross over
variety
how is the heteroduplex region repaired
gene conversion
depends on which strand is used as the template for repair
what’s the difference between horizontal and vertical cleavage
horizontal does not produce recombinants
process of recombination stage 1
- enzymes generate double-stranded break and single-stranded ends on paired homologous chromosomes
- this allows one DNA strand to invade other strands to match up
- separation of DNA in one strand runs along the sequence
- single strands find homologous DNA by DNA synapsis
stage 2 of dna recombination process
produces different outcomes
- release of invading strand
- capture of the second strand
what is the release of invading strand in dna recombination
additional DNA synthesis
ligation occurs
no cross over
what is the capture of second strand in dna recombination
- synthesis of heteroduplex region - branch migration
- Additional DNA synthesis occurs
- formation of cross arrangement where DNA crosses over between two strands, known as Holliday junction
what happens in the holliday junction
- an open form of cross occurs if the junction is rotated
- branch migration
- cross can be cut horizontally or vertically to separate chromosomes
what is branch migration
bases pair up
dna strands are swapped between chromosomes
which creates heteroduplex regions
minor base sequence differences result in region of DNA with low percentage of mismatch basepairs
what happens when the holliday region is cut horizontally?
- ligation occurs at joint adjacent ends
- after they join, strands straighten back out
- small region of variation
- no recombinant
what happens when the holliday region is cut vertically?
- ligation occurs at joint adjacent vertical ends
- strands straighten
- all strands crossed over
- recombination
how is RecA protein involved in recombination?
enables DNA single strand to pair with a homologous region of DNA double helix
when does recombination occur
meiosis
homologous recombination repair
features of recombination in meiosis
swapping over of chromosomes
4 haploid germ cells arent identical
unique due to cross over
hot spots occur - hybrid of genes
models of recombination
meselson radding
double strand break
features of the meselson radding model
- one strand cut
- DNA synthesis
- hanging strand displaces a strand from homologous duplex
- removal of displaced
- ligation
- holliday junction
features of double strand break
- loss of material from both strands
- two free sections of DNA
- two holliday junctions
