Chapter 13 lecture (Homologous recombination) Flashcards
why is Homologous recombination Essential in bacteria and in eukaryotic mitosis
to repair DNA damage and stalled replication forks
why is homologous recombination essential in eukaryotic meiosis
for generating diversity and for chromosome segregation
Recombination enzymes can use … as substrates
any pair of identical or nearly identical sequences
how is homologous recombination initiated
a double stranded break
in homologous recombination, how is the DbSB enlarged?
exonuclease will Degrade the 5’ end of both strands flanking the gap to form 3’-single stranded overhangs
A 3’ overhang will perform a … of the other duplex to make …
stand invasion
heteroduplex DNA
what will be used as template to synthesize new DNA through DbSB region
undamaged copy of DNA
after the DNA is synthesized in homologous recombination, what happens next?
The invading strand is released and base pairs with the single strand from the original duplex
in homologous recombination, High fidelity DNA polymerases will
fill in remaining gaps and all strands will be ligated
If it occurs after S phase
there will be identical sister chromatids to use as a template
If it occurs in G1,
it will have to use a non-identical homologous chromosome as a template
Meiotic recombination uses a homologous sequence as a template, even though
a sister chromatid is present
what is a key component of the homologous recombination mechanism in E. coli
RecA protein
RecA protein is similar to what in eukaryotes
Rad51
RecA binds to
the single stranded 3’ overhang produced after DbSB
RecA forces ssDNA into a
“double stranded” helical conformation
The RecA/ssDNA complex can displace
single strands from duplexes
If RecA/ssDNA finds a complimentary sequence of >15 nt
Complimentary strand will pair with ssDNA to form heteroduplex
Homologous recombination is necessary in eukaryotes
for
DNA repair
Restarting of collapsed replication forks
Needed for successful pairing of homologous chromosomes in prophase I
failure of homologous pairing leads to
nondisjunction in anaphase 1
Meiotic recombination begins in the same way as with general homologous recombination, except
The DbSB is induced by the Spo11 protein complex
The undamaged copy that is used as a template is a nonidentical homologous chromosome
what two proteins coat ssDNA overhangs and promote strand invasion
dmc1 and rad51
dcm1 is only active in
meiosis and has specificity for homologous chromosome
Prevents invasion of sister chromatid
rad51 is active
throughout the cell cycle for all DsDB recombination in eukaryotes
Loaded onto the ssDNA by the Brca2 protein
Loss of Brca2 function leads to reduced DsDB repair
Spo11 protein produces the
DbSB needed for meiotic recombination
how does Spo11 protein produce the DbSB needed for meiotic recombination
A tyrosine side chain of Spo11 attacks the phosphodiester backbone of DNA and creates a covalent enzyme-DNA complex
Energy of phosphodiester linkage is stored in enzyme-DNA linkage
DbSB is reversible and DNA strands can be easily resealed if recombination does not proceed
Sequence specificity of Spo11 activity is … but timing is …
low
highly regulated
Nuclease activity of Spo11 is related to existence of
recombination hotspots and coldspots in genome
hotspots
open active chromatin
promotors
coldspots
centromeres, telomeres
constitutive heterochromatin
holliday junction
where two homologous helices are held together by the exchange of two of the four strands
90% of the time, the DbSB are resolved as
non-crossover (patch) products
10% of the time, the DbSB are resolved as
The second strand is captured
Another Holliday junction is formed
The strands are cut and resolved as crossover (splice) products
what end of the strands are used as primers to extend the heteroduplexes
3’
When the extended single strands reaches the end of the gap, the single strands are ligated and
the second holiday junction is formed
The Holliday junction can move along the DNA using
branch migration
Upon resolution of both Holliday junctions using cleavage at different sites,
crossover products will be produced
Crossover control
The small number of crossovers on a chromosome will inhibit the resolution of nearby DbSB as crossovers
Even if a DbSB is resolved as a non-crossover a small heteroduplex patch remains that is a potential site of
gene conversion
gene conversion
One allele converted into another
An additional way to produce genetic variation in meiosis
Is a possible result of any DbSB-coupled homologous recombination
Including DNA repair mechanism
How does DbSB-coupled repair lead to gene conversion?
A polymorphic region is located inside the region that is involved in the strand invasion synthesis
The original allele will be replaced by one from the homologous duplex
If the homologous template is not identical, then it will create a heteroduplex mismatch
The heteroduplex mismatch randomly repaired by mismatch repair enzymes
Alters genotypic proportions of gametes
