Transcript Biochemistry 6/e

Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 28
DNA Replication, Repair, and
Recombination
Part III: DNA repair and recombination
Copyright © 2007 by W. H. Freeman and Company
(RNA primer?)
1. Cell death or transformation
2. Mutation inheritance
3. Replication stop
Types of DNA damage:
1.
2.
3.
4.
5.
Mismatches
Insertions or deletion (frame-shift)
Chemical modification of bases
Covalent cross-links
Backbone breaks
DNA replication-induced
Base substitutions:
A-T
T-A
A-T
T-A
C-G
G-C
C-G
G-C
Transition
Transversion
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: 36-82 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
C
G*
A
G*
A
A
T
2. deamination
A
T
A*
C
A*
C
C
G
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)
Interacts with SSB
3‘5’ proofreading
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)
CU
(spontaneous
deamination)
Uracil repair
AP site
(apurinic or
apyrimidinic)
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
DNAXrepair
Gene mutations
Cancers
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
5‘OH
recombinase
5‘OH
Tyr
Cre
Topo I
• Structural conservation
• 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