Principles BIOC2603 of Molecular Genetics
Recombination Brian Wong [e-mail]
Genetic recombination: reassortment of genetic material
(A) Independent assortment (for genes on different chromosomes) (B) crossing over (for genes on the same chromosome)
Crossover is the breakage of the two DNA molecules at the same position and their rejoining in two reciprocal recombinations.
Independent assortment produces 50 percent recombinants
For linked genes, recombinant frequencies are less than 50 percent
Four types of recombination
1.
Homologous recombination (general recombination)
Requires extensive sequence similarity between the two recombining DNA Takes place anywhere along the DNA
A
X
B
X
C
a A a
b b B
c C c
2.
Site-specific recombination
Occurs between two very limited and specific DNA sequences on both of the recombining DNA The two parental recombination sites are conserved in a simple reciprocal recombination event
CD X A B
A
D
C
B
Four types of recombination
3.
Transposition
Involves interaction of the two ends of a transposable element with a random site on the same or another DNA molecule Results in a new position and/or proliferation of the transposable element
+
4.
Illegitimate recombination
Processes that require neither sequence homology or site specificity e.g. non-homologous end joining
Biological roles of recombination
To generate genetic diversity For repair and replication To control gene expression (genetic switches) Genome evolution Introduces mutations and may result in diseases
Part I Homologous Recombination
Homologous recombination involves breakage and reunion of genetic material
A B
X
recombination by breakage and rejoining: involve the physical exchange of genetic material between the two recombining DNA recombination by copy choice: recombination occurs during replication by copying partly from one parent and partly from the other
a A a
b b B
A
B
a A
b b
Homologous recombination involves breakage and reunion of genetic material
phage 1 h
15N, 13C
phage 2 cI X h+
15N, 13C
cI+ labeled
labeled
(Heavy)
(Heavy)
Infect bacteria in 14N, 12C (light) medium. Separate phage progenies based on the densities of DNA by using CsCl density centrifugation.
h h+ + cI+ cI
Heavy recombinants were identified consistent with the breakage and reunion hypothesis. The copy-choice hypothesis predicted all recombinants were labled with 14N and 12C (light).
Meselson M (1964) J. Mol. Biol. 9:734-745.
Homologous recombination involves breakage and reunion of genetic material
knob extra piece of DNA
Corn with unusual chromosome C, colored; c, colorless Wx, waxy; wx, starchy
Recombination between C/c and Wx/wx
Recombinants Cytological crossing-over accompanied by genetical crossing-over
Homologous recombination involves breakage and reunion of genetic material
one strand contains BrdU, appears dark both strands contain BrdU, appears light
mitosis Grow cells in the presence of BrdU. BrdU incorporated into DNA.
BrdU
Stain with Hoechst 33258 and Giemsa, sister chromatid with one DNA strand containing BrdU appear dark, sister chromatid with 2 DNA strands containing BrdU appear light.
no sister chromatid exchange
x
homologous recombination between sister chromatids
Stages of a Neurospora cross
In Neurospora, meiosis is followed immediately by mitosis so a total of 8 haploid spores are observed.
The linear meiosis of Neurospora
In Neurospora, meiosis is followed immediately by mitosis so a total of 8 haploid spores are observed.
Each spore gives the genetic information of individual DNA strand of the 4 sister chromatids before meiosis.
Analysing the segregation patterns of the spores help to understand the molecular mechanism of homologous recombination.
chromosome
First meiotic division
Second meiotic division
Postmeiotic mitosis
Crossing-over occurs after DNA duplication
Crossing-over occurs after DNA duplication
Recombination in fungi
In a cross between a wild-type (A) and a mutant (a) allele, majority of the spores segregate in normal 4:4 ratio (no recombination); reciprocal recombination is also observed.
A A A A a a a a A A A A a a a a
mitosis
A A A A a a a a A A a a = a a A A
4:4 ratio: no recombination between centromere and the marker locus.
Product of A allele Product of a allele
A A A
A A a a A A a a
X
A a a a a
mitosis
A A a a A A a a
a a A A = A A a a
a a A A = a a A A
One recombination event between centromere and the marker locus.
Recombination in fungi
In addition to asci with 4:4 (A:a) ratio, non-Mendalian ratios 5:3, 3:5, 6:2, 2:6, and aberrant 4:4 segregation patterns were also observed. Gene conversions often associated with crossing-over of closely linked flanking markers. Explanations?
A A A a A a a a A A A a a a a a A A A A A a a a A A a a A a a a A A A a A A a a A A a a a a a a A A A A A A a a
aberrant 4:4
3:5
5:3
aberrant 5:3
2:6
6:2
Gene conversion: unidirectional transfer of genetic information
Models of homologous Recombination Holliday model (1964) Meselson-Radding model (1975) Double-strand break repair model (1983)
Holliday model of homologous recombination
Recombination is initiated by nicking of two homologous duplex DNA at the same site on strands with the same polarity
Broken strands separated from the original duplex DNA Reciprocal strand invasion: each nicked strand pairs with the complementary strands in the homologous duplex DNA
Holliday model of homologous recombination
Ligation creates a structure in which 4 strands of two duplex DNA are linked via a crossed structure (Holliday junction)
Holliday model of homologous recombination
Branch migration: movement of the Holliday junction generates heteroduplex (symmetric heteroduplex, i.e. heteroduplexes on both recombining DNA molecule)
Holliday model of homologous recombination
Isomerization of the Holliday junction to give a branch structure with 4 sectors of single-stranded DNA
Holliday model of homologous recombination
Holliday model of homologous recombination
Isomerization of the Holliday junction to give a branch structure with 4 sectors of single-stranded DNA
Holliday model of homologous recombination
Resolution of the Holliday junction to give non-crossover (flanking markers in parental configuration) and crossover (flanking markers exchanged) recombinant configurations.
Mismatch repair of heteroduplexes: either strands can be used as template
GATCCTATATATCTA CTAGGATATATAGAT
X
CTAGGATCTATAGAT GATCCTAGATATCTA
GATCCTATATATCTA CTAGGATATATAGAT or GATCCTATATATCTA GATCCTAGATATCTA CTAGGATCTATAGAT CTAGGATCTATAGAT Mismatch repair CTAGGATATATAGAT GATCCTAGATATCTA CTAGGATATATAGAT GATCCTATATATCTA Heteroduplexes or CTAGGATCTATAGAT GATCCTAGATATCTA
Holliday model of homologous recombination
A A A a A a a a A A A a A a a a
According to the Holliday model, reciprocal recombination results in the formation of two heteroduplexes
Mitosis before mismatch repair Mismatch repair in one of the heteroduplexes before mitosis Mismatch repair in both heteroduplexes before mitosis
A A A a A a a a
A A A a a a a a
A A A A A a a a
A A a a A a a a
A A A a A A a a
A A a a a a a a
A A A A A A a a
aberrant 4:4
3:5
5:3
aberrant 5:3
2:6
6:2
Holliday model needs to be modified
genetic experiments suggested that heteroduplex DNA often formed on only one chromatid, i.e. asymmetric heteroduplex (non-reciprocal recombination) was observed more often:
aberrant 4:4 segregations are diagnostic of symmetric heteroduplexes; they are rare as compared to 5:3 2 types of 5:3 segregation: normal 5:3 and aberrant 5:3. If there were symmetric heteroduplexes, one should expect equal proportions of the 2 types of 5:3 segregations but experiments showed that aberrant 5:3 are rare compared to normal 5:3.
Meselson-Radding model of recombination
a) b) Recombination initiated by a single-stranded nick on one duplex DNA. The 3 end of the nick serves as a primer for DNA synthesis, displacing a single-stranded DNA. The displaced strand then invades a homologous duplex DNA, forming a displacement loop (D-loop). The D-loop is degraded and the invading strand ligated to the recipient DNA. DNA synthesis on the donor duplex and degradation of the recipient duplex result in the extension of heteroduplex (asymmetric heteroduplex since only one duplex contains heteroduplex).
c)
d)
Meselson-Radding model of recombination
e) Ligation of the 5 and 3 ends results in the formation of a Holliday junction. The resulting Holliday junction can move by branch migration to form symmetric heteroduplex. Resolution of the Holliday junction gives either crossover or noncrossover configuration.
f)
Meselson-Radding model of recombination has an asymmetric and a symmetric heteroduplex phase.
A
A A A A A a a a
A A A A a a a
If the marker is on the asymmetric heteroduplex region: Mitosis before mismatch repair of heteroduplex The asymmetric phase explains why there were more 6:2 segregations and normal 5:3 segregations observed in recombinations.
A A A A A a a a
Mismatch repair in heteroduplex before mitosis
A A A A A A a a
5:3
6:2
Meselson-Radding model of recombination has an asymmetric and a symmetric heteroduplex phase.
A
A A A a A a a a
A A a A a a a
When the marker is on the symmetric heteroduplex region, its essentially the same as the Holliday model: mitosis before mismatch repair: aberrant 4:4 mismatch repair in one of the heteroduplex: 5:3, 3:5 mismatch repair in both heteroduplex: 2:6, 6:2
Meselson-Radding model of recombination
The models has an asymmetric heteroduplex and a symmetric heteroduplex phase. If markers are within the asymmetric heteroduplex region, then mismatch repair in the heteroduplex gives a 6:2 segregation pattern (gene conversion) or normal 5:3 segregation pattern if DNA replication occurs before mismatch repair. The asymmetric phase explains why there were more 6:2 segregation and 5:3 normal segregation observed. If markers are within the symmetric heteroduplex region, the aberrant 4:4, aberrant 5:3 and 6:2 segregation are explained by mismatch repair.
Homologous recombination
A A B
C C
Homologous recombination
A A
B B
C C
A
B
C
A
B
C
Double-strand break repair model of recombination
1. Recombination initiated by a double-strand break in one duplex. The break is enlarged to form a gap with 3 strand-stranded ends by exonucleases. 2. One of the free 3 end then invade a homologous duplex DNA, displacing a DNA strand and forms a D-loop 3. DNA repair synthesis from the 3 end of the invading strand enlarge the Dloop until the displaced strand anneals to the other free 3 end of the gapped duplex, forming asymmetric heteroduplex. 4. DNA synthesis from the second 3 end proceeds. Ligation of DNA result in the formation of 2 Holliday junctions. Branch migration can lead to formation of symmetric heteroduplex. 5. Resolution of the junctions give rise to recombinants with crossover or noncrossover configuration.
Double-strand break repair model of recombination
Double-strand break repair model of recombination
Double-strand break repair model of recombination
Double-strand break repair model
A A a a a a a a A A A a a a a a
marker within the gap
gap repair mismatch repair before mitosis
marker within asymmetric heteroduplex
Mitosis before mismatch repair
A A a a a a a a
A A A a a a a a
2:6
3:5
Double-strand break repair model
The gap are repaired by two rounds of single-strand repair synthesis and flanked by regions of heteroduplex. Two Holliday junctions are formed, branch migration of the Holliday junctions leads to formation of symmetric heteroduplex. Gene conversion (6:2 or 2:6 segregations) is the result of i) gap repair if the marker falls within the gap; ii) mismatch repair of heteroduplex if the marker falls within flanking asymmetric heteroduplex regions. 3:5 or 5:3 segregations results if mismatch in heteroduplex is not repaired.
Comparing the three models of recombination
Holliday model Meselson-Radding model single-strand break in one duplex asymmetric and symmetric 1 required Double-strand break repair model double-stranded break in one duplex asymmetric and symmetric 2 required initiation duplex is the recipient of information gap repair mismatch repair
Initiation
single-strand breaks in both duplex symmetric 1 not required
Heteroduplex No. of Holliday junctions DNA synthesis Exchange of sequence information Gene conversion
reciprocal exchange of initiation duplex is the information donor of information mismatch repair asymmetric strand transfer mismatch repair
The last step of homologous recombination is the resolution of Holliday junctions
Holliday junction is a crossover structure that links two recombining DNA Movement of the junction (branch migration) extend the length of heteroduplex. In E. coli, resolution of Holliday junction is carried out by RuvC resolvase.
crossover
non-crossover
Recombination proteins
Recombinase
RecA (E. coli) Rad51 (yeast)
Branch migration
RuvAB
Holliday Junction resolvase
RuvC (E. coli)
Endonucleases (to produce DNA strand breaks for initiation of recombination)
RecBCD (E. coli) Spo11 (yeast) I-SceI (yeast mitochondrial endonuclease encoded by intron of 21S rRNA gene)
Recombination Proteins in E. coli
RecA - promotes homologous pairing, strand exchange and formation of Holliday junctions. Binds to single-strand DNA to form a nucleoprotein filament that bind to double-strand DNA to search for homologous sequences. RuvAB - RuvA (22 kDa) binds to Holliday junctions and promote binding of RuvB to DNA; RuvB (37 kDa) catalyses branch migration; promotes branch migration of Holliday junctions in the presence of ATP, forming heteroduplex RuvC (19 kDa) - endonuclease that resolves Holliday junctions by introducing nicks into two DNA strands with the same polarity at an identical site; binds DNA substrate as a dimer in a sequence nonspecific manner but seems to cut DNA at preferred sites (5'-A/T TT G/C).
RecBCD Pathway in E. coli
RecBCD is made up of products from recB, recC, and recD genes, important for recombination and DNA repair RecBCD possess helicase activity, ssDNA exo- and endonuclease activity and dsDNA exonuclease activity, as well as ATPase activity Double-stranded DNA breaks (from ionization radiation, replication block etc) serve as initiation sites for homologous recombination RecBCD bind to end of a double-stranded break, the helicase activity then unwinds dsDNA and the ssDNA produced is cleaved , preferentially from 3 terminal
RecBCD Pathway in E. coli
When the DNA sequence (5-GCTGGTGG-3) is encountered, the 3 to 5 nuclease activity is attenuated while a 5 to 3 nuclease is activated. Therefore generating a ssDNA with the DNA sequence at the 3 end RecA protein load onto the single-stranded DNA, forming a nucleoprotein filament which direct the search of homologous sequences and strand invasion, resulting in the formation of Holliday junction. RuvAB is responsible for branch migration after the formation of Holliday junction: RuvA recognizes the Holliday junction structure and directs RuvB to the junction to catalyze branch migration RuvC is responsible for resolution of Holliday junction
Early steps in homologous recombination in E. coli
From: Kowalczykowski SC (2000) Trends in Biochemical Sciences 25:156-165.
Double-strand breaks initiate meiotic recombination
Recombination is initiated when Spo II enzyme makes double-stranded DNA cuts.
Processes involving homologous recombination Chromosome segregation
If crossing-over is prevented, chromosome fail to segregate properly.
Genome evolution Recombination-dependent DNA replication Recombinational repair of double-strand breaks
Repair damages induced by e.g. ionizing radiation Homing of homing endonuclease genes Programmed DNA rearrangement resulting in changes of gene expression e.g. mating type switching in yeast
Exon and gene duplication via unequal crossing over
Exon shuffling via recombination between interspersed repeats
Exon and gene duplication via unequal crossing over
Exon and gene duplication via unequal crossing over
Recombination-dependent Replication in Phage T4
Genome of phage T4 is a linear doublestranded DNA that are circularly permutated and terminally redundant. T4 replicates its DNA using both replication and recombination Origin-dependent replication of T4 chromosome produces daughter molecules with single-stranded 3' ends. The single-stranded end then invades other daughter molecule by homologous recombination in the presence of T4 uvsX gene product. The invading 3' end serves as primer for DNA synthesis. The displaced strand also serves as template for DNA synthesis.
T4 genome. DNA sequence represented by letters.
Model for recombinationdependent replication.
Kreuzer KN (2000) Recombination-dependent DNA replication in phage T4. TIBS 25:165-173.
Recombinational Repair of a Collapsed Replication Fork
Recombinational Repair of a Collapsed Replication Fork
Homing Endonuclease Genes (HEGs)
HEGs encode sequence-specific endonucleases. The recognition sequence (RS) is 15-30 bp long and usually occurs once in the genome. HEGs are located in the middle of their own recognition sequences. Most HEGs are encoded by self-splicing introns (group I & II) and inteins. Inteins are internal protein fragments produced from protein splicing and usually contain endonuclease and splicing activities. The allele without the HEGs are cleaved by the homing endonuclease and the double-strand break are repaired by homologous recombination (gene conversion) using the allele containing HEGs as template. Both chromosomes will contain the HEGs after repair.
From: Burt A and Koufopanou V (2004) Current Opinion in Genetics & Development 14:609-615.
Practical applications of homologous recombination Genetic mapping
Determine the order of genes in a chromosome Recombination frequency as a measure of distance between two markers
Gene targeting
Modify a chromosomal gene with a cloned copy of the gene
Recombination mapping
In a testcross (a heterozygote cross with a homozygous recessive), the recombinant frequency is proportional to the physical distance between two gene loci: the chance of crossing-over between two gene loci to produce recombinants is proportional to the physical distance between the two loci. A recombinant frequency of 1% is defined as 1 recombination map unit (m.u.) or centimorgan (cM). In human, 1 cM approximately equal to 1 million base pairs. A three-point testcross involving three loci can reveal the order of the loci and the distance between the loci.
e.g. (# of progeny) 252 243 250 248 recombinant frequency = 498/993 = 50%
e.g. (# of progeny) 236 322 10 10 recombinant frequency = 20/578 = 3.5% = 3.5 m.u.
Recombination between unlinked genes by independent assortment, producing recombinant frequency of 50%.
Recombination between linked genes by crossing over, producing recombinant frequency of less than 50%. In general, recombinant frequency between two gene loci indicates the physical distance between the two gene loci. 1 genetic map unit (m.u.) or centimorgan (cM) = 1% recombinant frequency.
Gene targeting by homologous recombination. A: insertion vector method. B: Replacement vector method.
Strachan T and Read AP (1999) Human Molecular Genetics 2. BIOS Scientific Publishers Ltd. London.
Reference Griffiths A.J.F. et al (2000) Introduction to Genetic Analysis. 7th ed. Freeman and Company, New York. Chapters 5,19. or Griffiths A.J.F. et al (2002) Modern Genetic Analysis: Integrating Genes and Genomes. 2nd ed. Freeman and Company, New York. Chapter 6. Watson J.D. (2004) Molecular Biology of the Gene. 5th ed. Pearson/Benjamin Cummings, San Francisco. Chapter 10. Leach D.R.F. (1996) Genetic Recombination. Blackwell Science, Oxford.