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

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.

Concept for chapter 14 & 15

Fig14.1 Two crossovers result in 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.

Overview of Recombination

http://engels.genetics.wisc.edu/Holliday/holliday3D.html

Fig14.2 Homologous vs non-homologous recombination. [E.Coli]

[site-specific recombination]

Specific recognition protein

Rarer than HR(HR)

Molecular Basis of Homologous Recombination

Fig 14-3. Formation of a crossover.

Crossover due to base homology may occur in DNA as 20-30bases, however, 50-100 bases is reasonable frequency.

heteroduplex:is any region of double-stranded nucleic acid (DNA, RNA), where the two strands come from two different original molecules.

Fig14-4. Rearrangement and Resolution of a Holliday Junction

RuvC, RecG act as resolvase.

Patch recombinants Short parch of heteroduplex remains in each molecule.

Formation of two hybrid DNA molecules by crossing-over

Fig 14-5. Migration of a Holliday Junction.

Bind to JunctionDrive migration

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

Fig14-6. RecBCD recognized Chi sites.

Immune response of E.coli (protect from foreign DNA)

Fig14-7. RecA promote strand invasion.

3’ tail

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 particleit circularizes upon entering bacterial cells& before integration

Fig14-8. Integration of Lambda DNA-overview.

att = attachment site

INT = integrase

O = center core of 15 bases = the same in phage & bacterial

B,P = different in size and sequence in bacterial & phagedsDNA

XIS = Excisionase

The control of INT & XIS activity determines it latency or not.

Fig14-8. Integration of Lambda DNA-Detail of crossover.

Fig14-10. Timeline of Eukaryotic Recombination in Yeast.

Eukaryotic recombination occurs in a span of ~2 hours.

resolution

• Recombination in Higher Organisms

Spo11 make dsb

Rad51 ~= RecA

Rad =response for recombination and repair

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 RSSpecific chemical defects.Lead to mutation.

Not included the synthetic enzymes and enzymes also used in normal DNA replication

• DNA Mismatch Repair System

Fig14-12. Principle of Mismatch Repair

Mismatch Repair Gap filled by DNA Pol III.

Note! most repair system using Pol I to replace short damaged region of DNA.

Cut out part of DNA strand containing wrong base.

Fig14-13. Methylated Bases-Chemical Structure.

Dam Protein (product of dam gene) DNA adenine methylase

Dcm Protein (product of dcm gene) DNA cytosine methylase

Recognition site is “Sequence-specific” & “Palindromic”

Not perturb base pairing

GATC CCTGG

Sequence unique for E.Coli

Fig14-14. Hemimethylated DNA

Palindrome make the DNA methylated equally on both strands.

Not perturb base pairing

[delay in fully methylation]1. During this period, many repair syste

ms check DNA.2. Control the initiation of new round of

bacterial DNA replication

Function of methylation Tell which is old, correct strand.

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

Fig14-15. MutSHL mismatch Repair System

L = hold togetherH = find the nearest GATC site & nick the non-CH3 strand

Pol III attach & repair the gap created by MutSHL 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)

Adenine Hypo-xanthineGuanine XanthineCytosine Uracil

deamination

Removal by DNA glycosylase (- bases)

Uracil-N-glycosylase(Ung protein)

Fig14-17. Removal of unnatural bases.

3’-OH

Pol I 1. recognizes the 3’-OH2. replaces a strench of ssDNA with AP site.

a-purine/ a-pyrimidine

Pol; 5’exonuclease

Fig14-18. dealing with oxidized guanine.

Prevent incorporation of preformed 8-oxoG into DNA.

MutT, MutM, MutY

• Specialized DNA repair mechanisms.

5-methylcytosine leads to mutational hot-spots.

Deamination of 5-methylcytosine:G T:G1. Occur spontaneously at any time and rarely during replication.2. Often goes unrepair3. 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

Fig14-19. Suicide demethylase for O-methyl bases.

O6-CH3-GO4-CH3-T

Fig14-20. Ada plays a dual role in removing alkyl groups

Ada = Adaptation to alkylation

Note! ~CH3 at N- and C- has different effects.

• Photoreactivation cleaves thymine dimersUvr excision repair system also PS:

Fig14-21. Photoreactivation cleaves pyrimidine dimers. No DNA synthesis

350-500nm

photolyase

Bind to dimer in darkbut lack energy to remove crosslink

• 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.

Fig14-22. Eukaryotic transcription-coupled excision repair.

helicase

Recruit the repair protein

Nick at the junction between ds and ssDNA.

• Repair by Recombination

Fig14-23. RecA and recombination repair.

TT dimer is still unrepaired in this process.

Old template is still damaged, but new made is correct.

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.

Fig14-25. DNA polymerase V is part of the SOS system. umu = UV mutagenesis

DNA pol V:

1.Subunits encoded by umu C and umu D2. lack of proofreading subunit3. Prefer GA rather than AA to pair TT dimer

For time to repair[no pol activity]

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 MutSBRCA1 (breast cancer A1) def breast & ovarian ca

• Double-strand Repair in Eukaryotes

by

Non-homologous End Joining

Fig14-26. Non-homologous End Joining in Mammals.

XRCC4 protein recruits DNA ligase IV to join two broken ends.

• 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.