Biochemistry Sixth Edition Chapter 28 DNA Replication, Repair, and Recombination Part III: DNA repair and recombination Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer

1.Cell death or transformation 2.Mutation inheritance 3.Replication stop (RNA primer?)

Types of DNA damage: 1.Mismatches 2.Insertions or deletion (frame-shift) 3.Chemical modification of bases 4.Covalent cross-links 5.Backbone breaks DNA replication-induced

Base substitutions: A-T T-A C-G G-C TransitionTransversion

DNA damage can be inherited by the future generations Ex. Huntington disease * Long tandem arrays of three nucleotide repeats * huntingtin: stretch of consecutive glutamines Wt: 6-31 CAG Disease: CAG (or longer)  array gets longer from one generation to next Cause?? alternative structure in DNA replication

Some other neurological diseases also have tri-nucleotide expansion polyglutamine  protein aggregation?

Post-replication DNA damages: Base-altering “mutagen”:

1. Reactive oxygen species  oxidation G  (oxidation) G* A G* A T GCGC

2. deamination A* C A* C G ATAT

3. alkylation G-C  T-A (transversion)

4. UV: covalent cross-link intrastrand x-link: Can’t fit in double helix interstrand x-link: DNA replication

5. Electromagnetic radiation (ex. x-rays)  single- and double-stranded breaks in DNA

DNA repair systems: 1.Recognize the offending base(s) 2.Remove the offending base(s) 3.Repair the resulting gap with a DNA polymerase and DNA ligase (using complementary strand)

3‘  5’ proofreading Interacts with SSB 1. Replication-coupled repair

Incorrect (weak) binding

2. Mismatch repair system MutS: recognition MutL: recruiting MutH MutH: endonuclease Methylation

3. Direct repair system DNA photolyase: Photoreactivating enzyme  Activated by light to cleave pyrimidine dimers

4. Base-excision repair Example #1: Uracil DNA glycosidase (Uracil repair) Example #2: AlkA (a glycosylase in E. coli, 3-methyladenine repair)

AP site (apurinic or apyrimidinic) Uracil repair C  U (spontaneous deamination) U vs. T in DNA

AlkA’s structure Glycosylase AP site (apurinic or apyrimidinic) AP endonuclease Deoxyribose Phosphodiesterase Polymerase/ligase

5. nucleotide-excision repair Repair intrastrand TT dimer 1 2 3

6. Double-strand break repair a.Nonhomologous end joining (NHEJ) ku70/80 b.Homologous recombination

DSB: *loss of genetic info. *chromosome translocation - ”hybrid” genes - incorrect expression

DNA repair Gene mutations Cancers X Genes for DNA-repair proteins  tumor-suppressor genes Xeroderma pigmentosum: skin cancer  Defective nucleotide-excision repair (UvrABC) Hereditary nonpolyposis colorectal cancer (HNPCC)  Defective DNA mismatch repair (1/200)  hMSH2 (MutS) and hMLH1 (MutL) p53: mutated in more than half of all tumors  Sensing double strand breaks  Activating repair systems or apoptosis

Cancer cells are sensitive to DNA-damaging agents Why? 1.They divide more rapidly 2.Defective DNA-repair

Ames test: detecting chemical mutagens (or carcinogens) Salmonella unable to grow revertants (can synthesize histidine) His- defective excision-repair systems addition of liver homogenate

DNA homologous recombination: important for replication, repair, and others

Homologous recombination (strand exchange): A DNA with a free end: Replication stop or double-stranded DNA breaks Many proteins involved One of the keys: RecA (AAA ATPase)

1.ssDNA invasion (strand invasion) *strand exchange; homologous sequence D-loop (displacement loop) New DNA synthesis

Case No. 1: DNA replication stop

No energy required!

Case No. 2: Double-strand break repair

Alternative resolution of Holliday structure 4-way Holliday junction

recombinase 5‘OH

Cre Topo I Structural conservation Tyr Tyrosine-DNA adduct Intra- vs. inter-duplex

DNA recombination: 1.Replication 2.Double-stranded break repair 3.Meiosis (meiotic recombination) 4.Antibody diversity 5.Virus infection 6.Gene knockout