Chapter 15 - Homologous and Site-specific Recombination Flashcards
Homologous Recombination definition
Exchange between similar (homologous) sequence of DNA.
In eukaryotes, common during meiosis.
Site-specific Recombination
Exchange between specific pairs of sequences. The two sites may or may not have same sequence (be homologous).
In prokaryotes, dif site to resolve catenated chromosomes.
Somatic Recombination
Exchange that occurs in non-germ cells that often leads to the generation of new genes.
Non-homologous Recombination (Recombination Repair)
Ligates blunt ends due to double strand breaks.
No crossing between the A and B gene loci in homologous recombination
= Only non-recombinant gametes
Crossing between the A and B gene loci in homologous recombination
= Both non- and recombinant gametes.
hotspot
Site in the genome at which the frequency of mutation (or recombination) is very much increased relative to neighboring sites.
Sister chromatid
Each of two identical copies of a replicated chromosome. As long as two copies remain linked at the centromere. Sister chromatids separate during anaphase in mitosis or anaphase II in meiosis.
Bivalent
The structure containing all four chromatids (two for each homolog) at the start of meiosis.
Synapsis
Association of the two pairs of sister chromatids (representing homologous chromosomes) that occurs at the start of meiosis.
Resulting structure is a bivalent.
Synaptonemal complex
Protein structure that forms between synapsed homologous chromosomes that is believed to be necessary for recombination to occur.
Chiasmata
Sites at which two homologous chromosomes have exchanged material during meiosis.
During the early part of meiosis,
homologous chromosomes are paired in the
synaptonemal complex.
The synaptonemal complex brings
chromosomes into juxtaposition.
Axial element
A proteinaceous structure around which the chromosomes condense at the start of synapsis; transition to the tripartite synaptonemal complex.
Lateral element
A structure in the synaptonemal complex that forms when a pair of sister chromatids condenses on to an axial element.
Central element
A structure that lies in the middle of the synaptonemal complex, along which the lateral elements
Cohesions
Connect sister chromatids so that they segregate
properly at mitosis or meiosis; form the lateral
elements.
Zip proteins
Transverse and link together to form the synaptonemal
complex; form the central element.
What initiates recombination?
Double-stranded breaks.
Spo11 protein
Initiates a double-stranded break during meiosis. 5’ ends are covalently bound to Spo11.
In mitotic cells, DSB occur most commonly
via DNA damage.
Exonuclease action generates
3’ single-stranded ends (or 3’-overhangs) that invade the other (donor) duplex.
D-loop
Displacement loop; loop of displaced DNA generated by strand invasion and extension during homologous recombination.
Double-strand breaks (DSB)
Breaks that occur when both strands of DNA duplex are cleaved at the same sites.
Initiates recombination.
(Also repair systems act on breaks created at other times).
5’-end resection
Generation of 3’ overhanging single-stranded regions that occurs via exonucleolytic digestion of the 5’ ends at a double-stranded break.
Single-strand invasion
The process in which a single-strand of DNA displaces its homologous strand in the duplex.
Heteroduplex DNA
DNA that is generated by base-pairing between complementary single-strands derived from the different parental duplex molecules; occurs during genetic recombination.
Gene conversion
Alteration of one strand of heteroduplex DNA to make it complementary with the other strand at any positions where there were mis-paired bases.
Also, the complete replacement of genetic material at one locus by a homologous sequence.
Holliday junction
An intermediate structure in homologous recombination in which the two duplexes of DNA are connected by the genetic material exchanged between two of the four strands (one from each junction).
Branch migration
The ability of a DNA strand partially paired with its complement in a duplex to extend its pairing by displacing the resident strand with which it is homologous.
Can occur in either direction when an unpaired single strand displaces a paired strand.
The exchange generates a stretch of heteroduplex DNA consisting of one strand from each parent.
Recombinant joint
The point at which two recombining DNA duplexes are connected by heteroduplex DNA and two Holliday junctions.
Splice Recombinant
DNA that results from a Holliday junction being resolved
by cutting the nonexchanged strands.
Patch Recombinant
DNA that results
from a Holliday junction being resolved
by cutting the exchanged strands.
MRN (MRX in yeast) complex - Function
Spo11 displacement and 5’-end resection. Chromosomal glue to prevent separation of DNA ends.
MRN (MRX in yeast) complexes contain
Mre11, Rad50, Nbs1
Rad50
Forms zinc-dependant dimer and connects to DNA ends to prevent separation of DNA ends.
Contains ATP binding/hydrolysis domains and forms a complex with Mre11 and Nbs1/Xrs2 (in yeast).
Mre11
Exonuclease action. Mutants are meiotic recombination deficient.
Nbs1 (Xrs2 in yeast)
Recruiting factor.
RecA
In E. coli, a DNA strand-transfer protein.
Assemble onto ssDNA to form helical nucleoprotein filaments in the presence of ATP.
ATP hydrolysis initiates strand invasion/assimilation.
The filaments hold the single strand in an extended conformation and promotes assimilation as long as one of the reacting strands has a free end.
RecA eukaryotic counterparts
Rad51 and Dmc1 proteins
A three-stranded intermediate
May be created when base pairing occurs between a ssDNA and its complement in a duplex molecule.
Then displacement of the corresponding strand of the duplex can occur.
Base pairing between the invading strand and its complementary strand in the donor requires:
- One of the DNA molecules must have a single-strand
- One of the DNA molecules must have a free 3’ end
- Single-stranded region and 3’-end must complementary
Ruv proteins
In E. coli, are involved in the stabilizing and resolving the Holliday junction.
RuvA
Recognizes the Holliday junction; binds all 4 strands by forming a tetramer structure.
RuvB
Helicase that catalyzes branch migration via ATP-hydrolysis.
RuvC
Endonuclease that resolves the recombinant intermediates in the Holliday junction.
ATTG tetranucleotide sequence
Hotspot for RuvC endonucleatic activity.
May determine which strand is targeted by RuvC for cleavage.
Hence may determine whether the Holliday junction is resolved by patch vs. splice recombination.
“Resolvasome” complex
Includes enzymes that catalyze (1) branch migration and (2) junction-resolving activity.
Unlike in prokaryotes, less is known about the resolution of the Holliday junction in eukaryotes.
Mus81
Mutants are defective in recombination.
Endonuclease that resolves Holliday junction in eukaryotes.
Similar to RuvC.
Supercoiling
The coiling of a closed duplex DNA in space so that it crosses over its own axis.
Positive supercoiling
Over-winding
Negative supercoiling
Under-winding
DNA Topoisomerase
Enzymes that catalyze DNA topological changes by transiently breaking 1 or 2 strands of DNA, passing 1 strand through the break, and resealing the DNA.
Covalently linked to DNA at the break.
Can act on any sequence of DNA.
Type I Topoisomerase
Break one (1) strand of duplex DNA. Recognize partially unwound segments of DNA; pass one strand through a break made in the other. Bonds are conserved; therefore, no input of energy is required.
Type II Topoisomerase
Break both (2) strands of duplex DNA. Can pass a duplex DNA through a double-strand break in another duplex. ATP is required to complete the reaction and reseal the break. Inhibiting the ATPase activity of the Type II results in a “cleavable complex” that contains broken DNA.
Gyrase
Type II DNA Topoisomerase that introduce negative supercoiling.
Reverse gyrase
Type II DNA Topoisomerase that introduce positive supercoiling are
Four types of eukaryotic Topoisomerase
IA, IB, IIA, IIB.
Eukaryotes DNA Topoisomerase enzymes function
Ensures replication fork movement, relaxes supercoils generated by transcription, and unlinks entangled chromosomes following replication (analogous to prokaryotes).
Group A Topoisomerase
Links the 5’-phosphate of broken DNA strand.
Group B Topoisomerase
Links the 3’-phosphate of broken DNA strand.
Recombinase
Enzyme that catalyzes site-specific recombination.
Phage lambda
In site-specific recombination, integrated (by reciprocal recombination between attP and attB) into the
bacterial chromosome.
Codes for an integrase that catalyzes the integration reaction.
Integrated between a site on the phage and the attachment (att) site on the E. coli chromosome.
Excised from the chromosome by recombination between the sites at the end of the linear prophage.
Core sequence
In site-specific recombination, the segment of DNA that is common to the attachment sites on both the phage
lambda and bacterial genomes.
The location of the recombination event that allows
phage lambda to integrate.
Integrase
Phage lamba codes for an integrase enzyme that catalyzes the integration reaction between the phage lambda and site of the E. Coli chromosome by breaking the phosphodiester bond and linking to the broken 3’-end.
Conserves energy.
Related to topoisomerase.
Cre
A recombinase.
Catalyzes a site-specific recombination between two identical lox sites, releasing the DNA between them.
A synapsed loxA recombination complex has a tetramer of Cre recombinases, with one enzyme
monomer bound to each half site.
The Cre/lox system is used extensively to create
“conditional knockouts” in mice, for example to delete a
gene of interest in a particular tissue type.
