DNA Repair and Recombination

Metabolism, radiation, environmental substances…

Liuh-yow Chen Molecular Biology of the , Chapter 5 Outline

1. DNA damages and

2. Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), etc.

3. Double-strand breaks repair: Nonhomologous End Joining (NHEJ) & (HR)

4. HR for Spontaneous Alterations of DNA

Hydrolysis Oxidation Methylation A/G T/C Depurination and Deamination

DNA lesions disrupt base pairing UV induces Pyrimidine Dimer

UV light triggers a oxidation reaction to generate a covalent linkage between two neighboring pyrimidines

Cyclobutane Pyrimidine dimers: TT, CC, CT, TC

Pyrimidine dimer disrupts base pairing Damages to Mutations

-> Mutations Damages-> Disrupted base pairing -> Repaired Base Excision Repair (BER)

• Base Damages, Single Strand Breaks (ROS attacks).

Recognition & cut

AP site: apurinic/ apyrimidinic Glycosylases

, xanthine

DNA methylation for inactivation Cytosine methylation-- deamination--Thymine C:G>T:G mispair Thymine DNA glycosylase (TDG), methyl-CpG-binding protein 4 (MBD4) Nucleotide Excision Repair (NER)

*Bulky lesions, UV, Tobacco smoke…

DNA adducts

ERCC1/XPF

* Recognition ? UV-DDB, RPA, XPD..

~ 30 nt humans Mismatch Repair (MMR)

Mismatches: base-base mismatches, / loops (IDLs) Recognition: MSH2/MSH6, MSH2/MSH3 Strand discrimination:MLH1/PMS2, MLH1/PMS1 Translesion Synthesis (TLS)

• TLS is used upon heavy damages (pyrimidine dimers) with DNA PCNA Ubiquitination replication.

• Translesion polymerases (TLS) are less discriminating in choosing nucleotide for synthesis and lack activity. > Error prone Double Strand Break (DSB)

p The most deleterious form of DNA damage p Generated by IR radiation, free radicals, topoisomerase II inhibitor, VDJ recombination, meiotic recombination etc p Repaired by two major pathways: Homologous recombination (HR) Nonhomologous end-joining (NHEJ) Double-Strand Break Repair

(G1/S/G2) (S/G2)

Error-prone Error-free NHEJ

Ku70/Ku80

DNA-PKc

Artemis nuclease

XRCC4/Ligase IV HR

* After DNA replication (S/G2)

* DSB recognition by MRE11/RAD50/NBS1

* End processing and resection: CtIP, MRE11/RAD50/NBS1, ExoI

* RecA/Rad51 mediate strand invasion RecA/Rad51 for Strand Invasion

/Rad51 RPA

BRCA2

Helical nucleoprotein filament

Rad52, Rad54

Homology searching HR Rescues Broken DNA Replication Forks DDR Activates Cell Cycle Checkpoints

G1 check point: Preventing DNA synthesis that allows damages to become mutations. S phase delay: Decreasing the rate of DNA synthesis G2 check point: Preventing division of the cells with damages ( breakage, loss). Diseases Due to Defects in DNA Repair HR for during Meiosis

Germ cell Genetic Recombination: Homologous pairing Generation of a novel set of genetic information that can be passed on from the parents to the offspring. Meiosis I HR / crossing over

Meiosis II

Gametes Crossover and

Genetic recombination = Gene conversion+ Chromosomal crossover HR in meiosis

Spo11- Meiosis specific endonuclease

Rad51 HR in meiosis

A B a b

A b a B Heteroduplex Holliday Junctions

* is a branched nucleic acid structure that contains four double-stranded arms joined together.

* Intermediate in meiosis.

* Isomerization: Structural transformation.

* Branch Migration: Breaking and re-forming of base pairs (ATP hydrolysis) Heteroduplexes formed by HR HR Results in Gene Conversion

!! HR results in genetic recombination (mutations) in meiosis !! !!! HR for DSB does not lead to gene conversion!!! Abstract | Mitochondrial DNA (mtDNA) faces the universal challenges of maintenance: the accurate replication, transmission and preservation of its integrity throughout the life of the organism. Although mtDNA was originally thought to lack DNA repair activity, four decades of research on mitochondria have revealed multiple mtDNA repair pathways, including base excision repair, single-strand break repair, mismatch repair and possibly homologous recombination. These mtDNA repair pathways are mediated by enzymes that are similar in activity to those operating in the nucleus, and in all cases identified so far in mammals, they are encoded by nuclear .