Download Recombination and Repair

Recombination and Repair
Chaper 14
高雄醫學大學
生物醫學暨環境生物學系
張學偉 助理教授
Concept for chapter 14 & 15
Homologous Recombination
 occur between any two highly similar regions of DNA,
regardless of the sequence
Non-homologous (Site-Specific) Recombination
(SSR)
occur between two defined sequences elements.
Transposition (Tn)
 occur between one specific seq and non-specific DNA
sites.
Overview of Recombination
In all cases of recombination, two
DNA molecules are broken and
rejoined to each other forming a
crossover.
Single crossover usually forms
short-lived hybrid DNA molecules.
promoter recombination of linear
chromosomes.
cannot cause recombination
between two circular DNA
molecules.
Double crossovers forms
recombination.
Fig14.1 Two crossovers result in recombination.
http://engels.genetics.wisc.edu/Holliday/holliday3D.html
[site-specific recombination]
(HR)
Rarer than HR
Specific
recognition
protein
Fig14.2 Homologous vs non-homologous recombination. [E.Coli]
Molecular Basis of Homologous Recombination
Crossover due to base
homology may occur in
DNA as 20-30bases,
however, 50-100 bases is
reasonable frequency.
Fig 14-3. Formation of a crossover.
heteroduplex:
is any region of doublestranded nucleic acid (DNA,
RNA), where the two
strands come from two
different original molecules.
Patch recombinants
 Short parch of heteroduplex
remains in each molecule.
RuvC, RecG act as resolvase.
Formation of two hybrid DNA
molecules by crossing-over
Fig14-4. Rearrangement and Resolution of a Holliday Junction
Bind to Junction
Drive migration
Fig 14-5. Migration of a Holliday Junction.
5 key steps in Homologous recombination
(i) alignment of 2 homologous chromosomes
(ii) introduction of breaks in DNAs
(iii) formation of initial short regions of base pairing between
the two recombining DNA molecules (strand invasion)
(iv) movement of Holliday junctions by repeat melting and
formation of base pair (branch migration)
(v) cleavage (or resolution) of Holliday junctions
Single-strand invasive and Chi sites
5’-GCTGGTGG-3’ Chi sites
naming
Immune response of E.coli
(protect from foreign DNA)
Fig14-6. RecBCD recognized Chi sites.
3’ tail
Fig14-7. RecA promote strand invasion.
Where is the dsb appeared?
Bacterial is haploid.
[no HR in sexual reproduction]
Bacterial recombination occurs between resident bacterial
chrosome and shorter incoming DNA.
e.g, transformation, transduction, conjugation.
In transformation, a cell can absorb and integrate
fragments of DNA from their environment.
In conjugation, one cell directly transfers genes (e.g.,
plasmid) to another cell.
In transduction, viruses transfer genes between
prokaryotes.
DNA bacterial viruses
= bacteriophages
Conjugation
= plasmid-directed transfer of DNA from one cell to another.
Site-specific Recombination
(non-homologous recombination)
Phage DNA properties
 is linear inside the virus particle
it circularizes upon entering
bacterial cells& before integration
att = attachment site
O = center core of 15 bases
= the same in phage & bacterial
dsDNA
B,P = different in size and sequence in
bacterial & phage
XIS = Excisionase
INT = integrase
The control of INT & XIS activity
determines it latency or not.
Fig14-8. Integration of Lambda DNA-overview.
Fig14-8. Integration of Lambda DNA-Detail of crossover.
• Recombination in Higher Organisms
resolution
Eukaryotic recombination occurs in a span of ~2 hours.
Fig14-10. Timeline of Eukaryotic Recombination in Yeast.
Spo11  make dsb
Rad =
response for recombination and repair
Rad51 ~= RecA
Fig14-11. Spo11 promotes dsb (double strand breaks)
• Overview of DNA repair
Different repair enzymes deal with different DNA damages
included:
Overall distortion of DNA structure.
 Mismatched RS( more sensitive than ERS) &
Excision RS
Specific chemical defects.
Lead to mutation.
Not included the synthetic enzymes and enzymes also used in normal DNA replication
• DNA Mismatch Repair System
Cut out part of DNA
strand containing
wrong base.
Mismatch Repair Gap
filled by DNA Pol III.
Note! most repair system
using Pol I to replace short
damaged region of DNA.
Fig14-12. Principle of Mismatch Repair
Not perturb base pairing
Dam Protein (product of dam gene)
 DNA adenine methylase
Dcm Protein (product of dcm gene)
 DNA cytosine methylase
Recognition site is “Sequence-specific” & “Palindromic”
GATC
Fig14-13. Methylated Bases-Chemical Structure.
CCTGG
Sequence unique for E.Coli
Palindrome make the DNA
methylated equally on both strands.
Not perturb base pairing
[delay in fully methylation]
1. During this period, many repair
systems check DNA.
2. Control the initiation of new round of
bacterial DNA replication
Function of methylation
 Tell which is old, correct strand.
Fig14-14. Hemimethylated DNA
The major mismatch repair system of E.Coli
is MutSHL.
Consist of MutS, MutH, MutL (proteins)
Note! Genes are mutS, mutH, mutL
(寫法不一樣)
mut = mutator,
def in mut  high mutation rate
Pol III attach & repair the gap
created by MutSHL system.
L = hold together
H = find the nearest GATC site &
nick the non-CH3 strand
Fig14-15. MutSHL mismatch Repair System
• General Excision Repair System
(“Cut and Patch” Repair)
1. The most widely distributed sysytem for DNA repair.
2. Recognize the bulge of DNA strand. e.g., UV (TT dimer)
3. Defect  UV sensitive (uvr = UV resistence)
4. Not detect mismatches, base analogs, certain methylated
bases.
Fig14-16. UvrABC Excision Repair System
Helicase
Single strand
Pol; 5’exonuclease
Nick are closed by DNA ligase
• DNA repair by
Excision of Specific Bases
(chemical changed bases,
CH3, O2)
deamination
Adenine  Hypo-xanthine
Guanine  Xanthine
Cytosine  Uracil
Removal by DNA glycosylase
(- bases)
Uracil-N-glycosylase
(Ung protein)
Pol I
1. recognizes the 3’-OH
2. replaces a strench of
ssDNA with AP site.
3’-OH
Pol; 5’exonuclease
a-purine/ a-pyrimidine
Fig14-17. Removal of unnatural bases.
Prevent incorporation of
preformed 8-oxoG into DNA.
MutT, MutM, MutY
Fig14-18. dealing with oxidized guanine.
• Specialized DNA repair mechanisms.
5-methylcytosine leads to mutational hot-spots.
Deamination of 5-methylcytosine:G  T:G
1. Occur spontaneously at any time and rarely during
replication.
2. Often goes unrepair
3. If occur at Dcm recognition site, it is repaired by “ very short
patch repair” (Vsr) system [nicking by Vsr endonuclease]
 Short length of strand remove by DNA pol I
O6-CH3-G
O4-CH3-T
Fig14-19. Suicide demethylase for O-methyl bases.
Note!
~CH3 at N- and C- has different effects.
Ada = Adaptation to alkylation
Fig14-20. Ada plays a dual role in removing alkyl groups
• Photoreactivation cleaves thymine dimers
PS: Uvr excision repair system also
350-500nm
photolyase
Bind to dimer in dark
but lack energy to
remove crosslink
Fig14-21. Photoreactivation cleaves pyrimidine dimers.
No DNA synthesis
• Transcriptional coupling of repair
Preferential repair of transcribed template DNA
strand.
Non-template strand is less likely to be repaired.
Bacteria:
Transcription-repair coupling factor (TRCF) can
detect a stalled RNA pol & direct UrvAB to block site.
helicase
Recruit the repair protein
Nick at the junction between ds and ssDNA.
Fig14-22. Eukaryotic transcription-coupled excision repair.
• Repair by Recombination
TT dimer is still unrepaired in this process.
Old template is still damaged, but new
made is correct.
Fig14-23. RecA and recombination repair.
SOS Error Prone Repair in Bacteria
 Allow DNA replication to proceed through severely
damaged zones, even at the cost of introducing mutations
[error prone repair]
Fig14-24. RecA and LexA control the SOS system.
DNA pol V:
1.Subunits encoded by umu C and
umu D
2. lack of proofreading subunit
3. Prefer GA rather than AA to pair
TT dimer
For time to repair
[no pol activity]
Fig14-25. DNA polymerase V is part of the SOS system. umu = UV mutagenesis
Like E.Coli, yeast, flies, and human all have
error-prone DNA polymerase.
In higher organisms, these repair enzymes
are more specialized and less error-prone.
Human error-prone pol, eta, can replicate
past TT dimer.
• Repair in Eukaryotes
Human MutS homologue = hMSH2 ~= E.Coli MutS
BRCA1 (breast cancer A1) def  breast & ovarian ca
• Double-strand Repair in Eukaryotes
by
Non-homologous End Joining
XRCC4 protein recruits DNA ligase IV to
join two broken ends.
Fig14-26. Non-homologous End Joining in Mammals.
• Gene conversion
Nonreciprocal step in DSB-repair sometimes result in
gene conversion.
Gene conversions are “not” associated with crossing
over.
Occur at Yeast mating-type switching
at Bacterial  genetic exchange via transduction or
conjugation
at eukaryote  homologous recombination in meiosis
Fig14-27. Gene Conversion Following Crossing over.
Comparison between gene conversion and DNA crossover. (a)
Two DNA molecules. (b) Gene conversion - the red DNA donates part of its
genetic information (e-e' region) to the blue DNA. (c) DNA crossover - the two
DNAs exchange part of their genetic information (f-f' and F-F').
An origin of gene conversion.
(a) Heteroduplexes formed by the resolution of Holliday structure or by other
mechanisms. (b) The blue DNA uses the invaded segment (e') as template to
"correct" the mismatch, resulting in gene conversion. (c) Both DNA molecules
use their original sequences as template to correct the mismatch. Gene
conversion does not occur.
Fig14-28. Mendelian ratios in Ascospore formation.