DNA Recombination and Repair Wenya Huang Medical Laboratory Science and Biotechnology

DNA Recombination and Repair

Wenya HuangMedical Laboratory Science and Biotechnology

National Cheng-Kung University

Outline

•DNA recombination•DNA damage•DNA repair•Assays for DNA damage and repair

3 ‘R’s of DNA metabolism:

Replication (copying of DNA prior to each cell division)

Recombination (exchanges between different DNA molecules in a cell)

Repair (restoration of altered DNA to its normal state)

recombination

Recombination in circular DNA

Cited from Genes VIII

e.g., phage integration into bacterial genome

attP

attB

Homologous Recombination

1. General (meiosis): chromosomal exchange between genes on the same locus. It occurs at the “four-strand stage of meiosis.

2. Specialized (site-specific) recombination: recombination between specific pairs of sequences. It is common in prokaryotes. E.g., phage DNA integration into bacteria, which include a short stretch of homology.

3. Transposition by transposons

Homologous recombination - Guided by base-pairing interactions between two homologous DNA molecules

•Two homologous DNA molecules in different chromosomes “cross over”. Their double helices break and the two broken ends join to their opposite partners to re-form two intact double helices, each composed of parts of the two initial DNA molecules.

•The site of exchange can occur anywhere in the homologous nucleotide sequences of the two participating DNA molecules.

•At the site of exchange, a strand of one DNA molecule becomes base-paired to a strand of the second DNA molecule to create a heteroduplex joint that links the two double helices. This heteroduplex joint can be thousands of base pairs long.

•No nucleotide sequences are altered at the site of exchange. The cleavage and rejoining events occur so precisely that not a single nucleotide is lost or gained.

Meiotic recombination – initiated by double-strand DNA breaks

•The process begins when an endonuclease makes a double-strand break in a chromosome. An exonuclease then creates two protruding 3’ single-stranded ends, which find the homologous region of a second chromosome to begin DNA synapsis. The joint molecule formed can eventually be resolved by selective strand cuts to produce two chromosomes that have crossed over.

Recombination in meiosis

Cited from Genes VIII

DNA synapsis

At DNA synapsis, the base pairs form between complementary strands from the two DNA molecules. This base-pairing is then extended to guide the general recombination process, allowing it to occur only between DNA molecules that contain long regions of matching DNA sequence.

Cited from Mol Biol. Cell, 4th edition

Homologous recombination guided by DNA double-strand break

Recombination

Cited from Genes VIII

•Double-strand break•Unwinding and trimming•Strand invasion (single-strand assimilation)

•Strand migration and displacement

•DNA synthesis

•resolutionCited from Genes VIII

Recombination In E. coli: RecABCD system-Activate RecBCD nuclease and helicase

-RecBCD nuclease and helicase

-- RecA

--DNA polymerase

-Branch migration (RecA and RuvAB)

-DNA polymerase

Cited from Genes VIII

Recombination in E. coli: RecABCDNuclease and helicase

Cited from Genes VIII

•RecBCD binds to DNA at a double-stranded end. Two of its subunits have helicase activities: RecD functions with 5’-3’ polarity, and RecB functions with 3’-5’ polarity. •RecD initially translocates along DNA and unwinds the double helix.•RecBCD degrades the released single strand with the 3’ end. When it reaches the chi site, it recognizes the strand of the chi site in single-stranded form. It cleaves the strand of the DNA at a position between four and six bases to the right of chi. •Recognition of chi site causes the RecD subunit to dissociate or become inactivated, the nuclease activity is then lost.•RecBC cointinues to perform the helicase activity.

Chi sequence

5’ GCTGGTGG 3’3’ CGACCACC 5’

Occur naturally in E. coli DNA per 5-10 kb

Stimulate recombination in its vicinity (< 10 kb)

Targets for the RecBCD complex

RecA (in E. coli)

•Binds to single-strand DNA to form a nucleoprotein filament.•It is also a DNA-dependent ATPase.•It associates much more tightly with DNA when it has ATP bound. •Each RecA monomer has more than one DNA-binding site to hold a single strand and a double helix together. It catalyzes a multi-step DNA synapsis reaction between a DNA double helix and a homologous region of single-stranded DNA. It helps formation of the three-stranded intermediate.•It enables a DNA single strand to pair with a homologous region of DNA double helix.•It catalyzes unidirectional branch migration, readily producing a region of heteroduplex DNA that is thousands of base pairs long.

RecA protein and DNA synapsis

RecA: Invading of DNA with free end

Cited from Genes VIII

RecA homologs in eukaryotes

•There are at least 7 RecA homologs in humans and mice. These include Rad51, Rad50, Rad52, BRAC1 and BRAC2, etc.

•Each homolog has its special catalytic activities and its own set of accessory proteins.

•Among these proteins, Rad51 plays the central roles. It is functionally most similar to RecA.

Branch migration

After DNA synapsis has occurred, an unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point without changing the total number of DNA base pairs.

Branch migration

Cited from Genes VIII

Branch migration

Cited from Genes VIII

Heteroduplex

RuvAB complex: branch migration

Cited from Genes VIII

RuvAB complex

•It processes the Holliday junctions into mature recombination products

•RuvA binds the Holliday junctions as a tetramer or double tetramer with high affinity and unfolds the junctions from the stacked X-structure.

•RuvB targets the Holliday junction by interaction with the RuvA-junction intermediate. Two hexameric rings of RuvB encircle opposing DNA duplex arms of the junction and act as ATP-dependent DNA motors extruding heteroduplex DNA.

Holliday junction

•In a Holliday junction, the two homologous DNA helices that have initially paired are held together by the reciprocal exchange of two of the four strands present, one originating from each of the helices.

Crossing over: Holliday junction

Holliday junction

Resolution of Holliday junction

Cited from Genes VIII

RuvABC in bacteria

Cited from Genes VIII

RuvC

•It is also called DNA resolvase

•It is a Holliday junction-specific endonuclease, which cleaves Holliday junction into two mature recombinant products

Homologous and non-homologous recombination pathways for double-stranded DNA breaks

Cited from Mol Biol. Cell, 4th edition

Homologous recombination (HR) takes place in M (meiosis) and late S–G2 (DNA repair) phases and involves the generation of a single-stranded region of DNA, followed by strand invasion, formation of a Holliday junction, DNA synthesis using the intact strand as a template, branch migration and resolution.

Non-homologous end-joining (NHEJ) takes place throughout the cell cycle and involves binding of the KU heterodimer to double-stranded DNA ends, recruitment of DNA-PKcs (officially known as protein kinase, DNA-activated, catalytic polypeptide (PRKDC)), processing of ends (including Artemis-dependent processing) and recruitment of the DNA ligase IV (LIG4)–XRCC4 complex, which brings about ligation.

Mammalian

Ku70, Ku80 and DNA strand ends

Cited from Genes VIII

Mre112Rad502Xrs2(Nbs1) pentamer

•It is named MRN (mammals) or MRX (yeast).

•The terminal CXXC-hooks form an interlocking dimerization interface in the Rad50 coiled-coils.

•Intermolecular dimerization between individual complexes allows MRX (MRN) complex to tether DNA ends and perhaps tether and align sister chromatids.

Rad50

• catalytic domain: intramolecular ATP-binding cassette (ABC) type ATPase domain. Rad50 forms homodimers in the presence of ATP. Dimerization creates a DNA-binding interface across the Rad50 dimer.

• Mre11 binds to the base of the coiled-coil near the Rad50 DNA-binding interface, suggesting the formation of a composite DNA-binding site within the (Mre11)2/(Rad50) 2 heterotetramer.

Mre11 nuclease

(a)3'-5' exonuclease activity on blunt and 3' recessed ends.

(b)endonuclease activity on circular and linear ssDNA.

(c) endonuclease cleavage of hairpin ends and 3' ssDNA overhangs at the single-/double-stranded transition.

(d)Rad50 and Xrs2 (Nbs1) both influence MRX substrate binding and potentiate the intrinsic nuclease activity of Mre11

The MRE11/RAD50/NBS1 complex

It is the central player in the cellular response to DNA double-strand breaks, including homologous recombination, non-homologous end joining and telomere breakage. The Mre11 complex possesses an ATP-stimulated nuclease to process heterogeneous DNA ends and long coiled-coil tails to link DNA ends and/or sister chromatids.

Xrs2 (yeast) or NBS1 (mammals)

Nbs1 directly interacts with Mre11. The MRN complexplays critical roles in initiation of the intra S-phasecheckpoint in response to DNA damage, and Nbs1 isphosphorylated as part of the damage signal by kinasesfrom the ATM family of protein kinases.

Functional or physical interactions of the MRN complex with other DNA repair/checkpoint proteins

DNA replication, transcription and recombination cause topological changes in DNA structures.

DNA replication and topology

Cited from Genes VIII

1.

2.

Resolution of topological changes of DNA molecules

Cited from Genes VIII

DNA Topoisomerase

•Relax or introduce supercoils in DNA

•Transiently break in one or both strands of DNA, passing the unbroken strands through the gap and then resealing the gap.

Type I topoisomerase – break one strand only

Cited from Genes VIII

Type II topoisomerase – break two strands

Cited from Genes VIII

DNA Gyrases: supercoiling circular DNA

Cited from Genes VIII

DNA Repair

DNA repair factors: tumor suppressors

1. MSH2 and MLH1 (DNA mismatch repair)Types of cancers: hereditary nonpolyposis colorectal cancer (Lync

h syndrome), endometrial, gastric, ovarian, bladder cancer

2. Ataxia telangiectasia mutated, ATM (DNA damage sensor, protein kinase)

Types of cancers: Ataxia telangiectasia, T-cell lymphoma, other lymphoreticular malignancies

3. Nijmegen breakage syndrome, NBS1 (recombination and DNA repair, S phase checkpoint control)

Types of cancers: Nijmegen breakage syndrome, T cell lymphoma, and other lymphoreticular malignancies

4. BRCA1, BRCA2 (recombinational DNA repair)Types of cancers: familial breast and ovarian cancer

5. Faconi anemia, FA (recombinational DNA repair, S phase checkpoint)Types of cancers: fanconi Anemia, acute myelogenous leukemia

DNA damage

1. Spontaneous (endogenous): oxidative stress, deamination, loss of nitrogen bases, uridine incorporation

2. Induced (environmental): X-ray and UV irradiation, benzene (smoking, air pollution), cisplatin (chemotherapy)

3. Mismatch: replication errors

Replication errors caused by UV irradiation

T T

A A

A G

A A

T<>T

+T TA A

A GT C

T<>T

transition

+

Deamination in nitrogen bases

Cited from Mol. Biol. Cell., 4th edition

Deamination

DNA repair1. Reversion repair (damaged nitrogen base is re

paired without removing it)• Methylguanine methyl transferase (MGMT) for methylated

DNA repair• Dioxygenase for methylated DNA repair• Photolyase for UV-induced T-T dimer repair

2 Excision repair (damaged nitrogen bases/nucleotides are removed then replaced with intact ones)

• Base excision repair• Nucleotide excision repair• Mismatch repair• Recombinational repair

Base excision repair (BER)

The BER pathway deals with smaller damage to individual bases, such as oxidation, methylation, depurination, and deamination.

Many BER lesions arise via a number of endogenous processes, the most common being spontaneous depurination, which is estimated to occur at a rate staggering of 104 events per human cell per day.

The BER pathway is initiated by a DNA glycosylase(the enzyme that cleaves nucleotide at glycosidicbond) that is capable of recognizing a particular subset of base alterations.

Glycosidic bond

DNA glycosylases

• They are small monomeric proteins (20 to 40 kDa) that function without additional cofactors and access nucleotides via ring flipping.

•DNA glycosylases fall into two categories, monofunctional and bifunctional depending on their catalytic properties.

•Monofunctional glycosylases cleave the glycosylic bond leaving an abasic site, while bifunctional glycosylases contain an additional AP-lyase activity creating a 3’-fragmented deoxyribose.

DNA glycosylases can be subdivided into 4 groups according to their main substrates:

•Excision of uracil (UNG, SMUG1)•Excision of uracil-containing mismatched (TDG, MBD4)• Excision of alkylated bases (ANPG or AAG)•Excision of oxidized bases (OGG1, MYH, NTG1, NTG2, NEIL1)

GlycosylaseMBD4

MPG

MYH

NEIL1

NEIL2

NEIL3

NTH1

OGG1

SMUG1

TDG

UNG

Substrate specificityU and T opposite G

3-MeA, 7-MeG, 3-MeG ethenoA, hypoxanthine

A opposite 8-OxoG

Formamidopyrimidines oxidised pyrimidines (e.g.

thymine glycol)

5-Hydroxyuracil; 5-hydroxycytosine

Fragmented and oxidised pyrimidines

Ring-saturated, oxidised and fragmented pyrimidines

8-OxoG paired with C, T and G

Uracil

U, T or ethenoC, opposite G T opposite G, C and T

Uracil

Human DNA glycosylases

Enzymes required forbase excision repair

DNA glycosylase

AP endonuclease &phosphodiesterase

DNA polymerase &ligase

AP (apurinic/apyrimidinic) endonuclease

It cleaves DNA sugar-phosphate backbone at the nucleotide where the nitrogen base is already cleaved by a DNA glycosylase.

DNA phosphodiesterase

It cleaves at phosphodiester bond on DNA and removes the AP nucleoside from the DNA molecule.

Nucleotide Excision Repair (NER)

DNA repair pathway which recognizes damaged regions based on their abnormal structure as well as on their abnormal chemistry, then excises and replaces them.

Steps in NER

1.Damage recognition 2.Binding of a multi-protein complex at the damaged site 3.Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5' and 3' sides 4.Removal of the damage-containing oligonucleotide from between the two nicks 5.Filling in of the resulting gap by a DNA polymerase 6.Ligation

Enzymes required fornucleotide excision repair

Nucelase

DNA helicase

DNA polymerase &ligase

E. coli NER: UvrABC excinuclease

E. coli UvrABC system

Nucleotide excision repair in mammals

1. transcription-coupled repair (TCR) (NER at the actively transcribed strand of the gene): It is more efficient than global genomic repair.

2. Global genomic repair (GGR) (NER at the nucleotides where is not actively transcribed)

TCR and GGR

•TCR and GGR are initiated by different NER factors for DNA damage recognition.

•TCR is initiated and signaled by stalling of RNA polymerase at the DNA lesion site; whereas GGR is initiated by recognition of the abnormal structure at the DNA lesion site.

Transcription-coupled repair

RNA polymerase stalling at DNA damage site

Global genomic repair

DNA damage recognition

Methylation and mismatch repair in E. coli

Cited from Genes VIII

Mismatch recognition: which strand is wrong?

1. Prokaryotes, based on methylation: the old (right) strand is methylated, the new (wrong) is not.

2. Mammalians, based on presence of single-strand break (SSB): the strand with SSB is the new (wrong) strand, the one with no SSB is the old (right) strand.

Mismatch repair in humans

Measurement of DNA damage/repair

1. Survival to genotoxic agents2. Comet assay3. Southwestern immunoblot4. Host cell reactivation assay5. In vitro DNA repair assay

Cell survival to UV irradiation

Comet assay to detect DNA lesions

Slide

Alkaline unwinding

Cell lysis

Electrophoresis

DNA staining

Cells

lesion-specific endonuclease, e.g., FPG

Comet assay scoring

type2

Type 0 Type 1

Type 4

Type 2 Type 3

0

50

100

150

200

250

300

0 μM 5 μM 10μM 20μM 50μM

DN

A d

amag

e in

dex

no fpg fpg digestion

Huh 7 cell treat with H2O2

DN

A d

amag

e in

dex

South-Western immunoblot

UV

cells

Incubation

genomic DNADenatured DNA in NaOH

DNA on N+ membrane

Anti-DNA lesion antibody Western blot

South-Western immunoblot

Host cell reactivation assayMeasurement of DNA repair activity in cells

Damaged pCMVLux

Huh 7 cells

Co-transfection

Luciferase kit

luminometer

p3A-SAg

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1

mock WT Deletion 1 Deletion 2

mock WT 1 2

Fol

d re

pair

In vitro NER assay