dna repair types direct repair –alkylguanine transferase –photolyase excision repair –base...
TRANSCRIPT
DNA Repair Types
• Direct repair– Alkylguanine transferase– Photolyase
• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair
• Recombination repair
N N
NN
O
NH2
CH3
O6-methylguanine
AGT-CH2-SH
N NH
NN
O
NH2
AGT-CH2-S CH3
Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)
In a stoichiometric reaction, the O6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded.
Direct repair: O6-alkylguanine DNA alkyltransferase (AGT)
AGT inhibitor O6-benzylguanine is in clinical trials to be used in conjunction with antitumor alkylnitrosoureas
N
NO
NHR'Cl
O
2-chloroethylnitrosoureas
CldG in DNA
O
N
N
N
O
NH2N
O
HO
HHHH
Cl
DNA-DNA cross-links
tumor cell death
AGT overexpression in tumors makes them resistant to alkylnitrosoureas
N
NO
NHR'Cl
O
2-chloroethylnitrosoureas
CldG in DNA
O
N
N
N
O
NH2N
O
HO
HHHH
Cl
DNA-DNA cross-links
AGT
normal dG
tumor cells survive
Combination therapy with O6-benzylguanine overcomes tumor resistance to alkylnitrosoureas
N
NO
NHR'Cl
O
2-chloroethylnitrosoureas
CldG in DNA
O
N
N
N
O
NH2N
O
HO
HHHH
Cl
DNA-DNA cross-links
tumor cell deathAGT
normal dG
N
N
N
O
NH2NH
Excision Repair
Takes advantage of the double-stranded (double information) nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
Antiparallel DNA Strands contain the same genetic information
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
A ::
G
T ::
T
A
3'
3' 5'
5'
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
Original DNA duplex DNA duplex with one of the nucleotidesremoved
Repaired DNA duplex
• Used for repair of small damaged bases in DNA (AP sites, methylated bases, deaminated bases, oxidized bases…)
• Human BER gene hogg1 is frequently deleted in lung cancer
Base excision repair (BER)
HN
NH
NN
O
NH2
O
8-oxo-G
OHOO
O
Abasic site (AP site)
NNH
NH
N
O
O
Xanthine
N N
NN
NH2
Me
N3-Me-Ade
Uracil DNA glycosylase removes deaminated C
N
N
NH2
O
NH
N
O
O
Uracil
BERC
Not normally present in DNA
No Me group
Cytosine
Thymine
H3CNH
N
O
O
Normal DNA baseNot recognized by BER
Nucleotide Excision Repair
• Corrects any damage that both distorts the DNA molecule and
alters the chemistry of the DNA molecule (pyrimidine dimers,
benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
5'
3'
NH
NH
NN
NO
HO
HOOH
HOO
OH
• Xeroderma pigmentosum is a genetic disorder resulting in defective NER
Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
1. Recognize parental DNA strand (correct base) and daughter strand (incorrect base)
Parental strand is methylated:
2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt)
N
N
NH2
O
H3CN N
NN
HNMe
G T
Genetic diseases associated with defective DNA repair
Xeroderma Pigmentosum NER
Hereditary nonpolyposis MMRcolorectal cancer
Cockrayne’s syndrome NER
Falconi’s anemia DNA ligase
Bloom’s syndrome BER, ligase
Lung cancer (?) BER
Chapter 3. RNA synthesis (Transcription)
Required reading: Stryer 5th edition p. 129-132, 781-792(or Stryer 4th edition p. 95-102, 841-849, 851-859)
Flow of genetic information
DNA RNA Proteins Cellular Action
transcription translation
DNA
rep
licat
ion
O
N
NN
N
NH2
O
OHO
PO
O
O-
NH
N
N
O
NH2N
O
OHHO
PO
O
O-
N
NH2
ON
O
OHO
PO
O
O-
NH
O
ON
O
OHO
PO
O
O-
NH
N
N
O
NH2N
O
OHOH
PO
-O
PO
O
O-
OP
O
-O
O-
RNA is a biopolymer consisting of ribonucleotide units
5’
3’
RNA is usually single stranded but can form hairpin structures
by folding over the same strand:
G
GC
U
U
5'-U•C•C•C•A•C A•U•U•U-3'
G•C A•U C•G C•G G•C C•G C•G G•C
Double-stranded
region(stem)
Loop
Structural differences between DNA and RNA
H3CNH
NH
O
O
Thymine (T)
NH
NH
O
O
Uracil (U)
DNA RNA
O
H
HHH
CH2
HO
HOBase
2'-deoxyribose
O
OH
HH
CH2
HO
HOBase
ribose
H
O
OH
O
BASE
1'
O
O
OH
OBASE3'
1'3'
2'5'
5'
2'
RNA Types
mRNA = Messenger RNA; an RNA copy of the DNA sequence (gene) used as a template for protein synthesis
tRNA = Transfer RNA; a small RNA that is attached to an amino acid which can be added to a growing peptide chain
rRNA = Ribosomal RNA; component of ribosomes with catalytic and structural function
snRNA = Small nuclear RNA, involved in RNA splicing ineukaryotes
RNA in E. coli
Type Relative amount Mass # of Nucleotides ____________________%_________________kDa_____________________
rRNA 80 23S 1.2X103 3700
16S 0.6X103 1700
5S 3.6X101 120
tRNA 15 2.5X101 75 mRNA 5 heterogeneous sn RNA <1
RNA synthesis
• RNA synthesis involves transcribing a specific portion of DNA strandinto RNA sequence
•RNA polymerases sequentially add ribonucleotides to the 3’ end of an RNA polymer using DNA strand as a template (5’ 3’ direction)
•RNA Pol selects ribonucleotides complementary to the DNAtemplate and catalyzes the formation of new phosphodiester bonds. This process is repeated as the enzyme moves along the DNA.
(RNA)n bases + NTP (RNA)n+1 bases +PPi
2Pi
RNA Synthesis: addition of new NTPsfollows Watson-Crick rules
HN
N
O
O
NN
N
NNH 2
A•U
NH
N
NO
NH 2
NN
N
H2N
O
G•C
Template base Incoming baseG CC GU AA U
The sequence of the RNA transcript is complementary to the transcribed DNA strand
and is the same as the coding strand
Coding strand
Non-coding (template) strand
5’…A T G G C C T G G A C T T C A…3’3’…T A C C G G A C C T G AA G T…5’
5’- A U G G C C U G G A C U U C A…3’
Transcription
Translation
…Met-Ala-Trp-Thr-Ser… Peptide