21
DNA Repair Types Direct repair Alkylguanine transferase Photolyase Excision repair Base excision repair Nucleotide excision repair Mismatch repair Recombination repair

DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

Embed Size (px)

Citation preview

Page 1: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

DNA Repair Types

• Direct repair– Alkylguanine transferase– Photolyase

• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair

• Recombination repair

Page 2: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch 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)

Page 3: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 4: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 5: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 6: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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.

Page 7: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 8: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

• 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

Page 9: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 10: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 11: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 12: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 13: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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)

Page 14: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

Flow of genetic information

DNA RNA Proteins Cellular Action

transcription translation

DNA

rep

licat

ion

Page 15: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 16: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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'

Page 17: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 18: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 19: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 20: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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

Page 21: DNA Repair Types Direct repair –Alkylguanine transferase –Photolyase Excision repair –Base excision repair –Nucleotide excision repair –Mismatch repair

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