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DNA Repair Types • Direct repair – Alkylguanine transferase – Photolyase • Excision repair – Base excision repair – Nucleotide excision repair – Mismatch repair • Recombination repair Direct repair: O6-alkylguanine DNA alkyltransferase (AGT) 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. O N N CH3 O N N N NH AGT-CH2-SH NH2 O6-methylguanine N N AGT-CH2-S NH2 CH3 AGT inhibitor O6-benzylguanine is in clinical trials to be used in conjunction with antitumor alkylnitrosoureas NO N Cl NHR' O 2-chloroethylnitrosoureas Cl O N Cl dG in DNA N O H H O O N NH2 DNA-DNA cross-links H H N H tumor cell death AGT overexpression in tumors makes them resistant to alkylnitrosoureas NO N Cl NHR' O 2-chloroethylnitrosoureas Cl O N Cl dG in DNA N O H H O O N N NH2 DNA-DNA cross-links H H H AGT normal dG tumor cells survive Combination therapy with O6-benzylguanine overcomes tumor resistance to alkylnitrosoureas NO N Cl NHR' O 2-chloroethylnitrosoureas Cl O N dG in DNA Cl N O H H O O N N DNA-DNA cross-links H H NH2 H O N N H AGT N N NH2 normal dG tumor cell death 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 5' 3' 5' 3' 5' 3' 3' A :: T A :: T A :: T G ::: C G G ::: C T :: A T :: A T :: A 5' Original DNA duplex 3' 5' DNA duplex with one of the nucleotides removed 3' 5' Repaired DNA duplex Base excision repair (BER) • Used for repair of small damaged bases in DNA (AP sites, methylated bases, deaminated bases, oxidized bases…) H N O N OH N NH NH O N 8-oxo-G NH2 O O O O NH2 Abasic site (AP site) N N H Xanthine O N N N N Me N3-Me-Ade • Human BER gene hogg1 is frequently deleted in lung cancer Uracil DNA glycosylase removes deaminated C No Me group NH2 O N N BER NH O N Cytosine C O Not normally present in DNA U rac il O H3C NH N Thymine O Normal DNA base Not 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). O H3C O NH N 5' O O O P OO N O HO32N CH Pt NH H2N N Cl Cl O O 3' • HO O NH H2N N OHNH O N 2 HOPt OH H2N OH2 HO OH H N NH2 N -GGH2N H2N N Pt N O N N NH N NH2 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: G T 1. Recognize parental DNA strand (correct base) and daughter strand (incorrect base) Me Parental strand is methylated: H3C NH2 HN N N N O N N N 2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt) Genetic diseases associated with defective DNA repair Xeroderma Pigmentosum NER Hereditary nonpolyposis colorectal cancer MMR 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 replication DNA RNA Proteins transcription translation DNA Cellular Action RNA is a biopolymer consisting of ribonucleotide units NH2 5’ O O O -O P O P O P OO- -O O N N N N NH RNA is usually single stranded but can form hairpin structures by folding over the same strand: N NH2 N NH2 U N O O O OH O P OO O H O OH O P OO O Loop N N U O N O O N 3’ O OH O P OO O N OH OH NH N NH2 5'-U•C•C•C•A•C G G G•C A•U C•G C•G G•C C•G C•G G•C O NH O OH O P OO O C Doublestranded region (stem) A•U•U•U-3' Structural differences between DNA and RNA DNA RNA O O H3C NH NH N O H Uracil (U) N O H Thymine (T) HO CH2 H HO O Base CH2 H O H H Base H H H H O H O 2'-deoxyribose O BASE 2' 5' O 3' O 1' H OH ribose O O 5' 3' BASE 1' O 2' OH 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 in eukaryotes RNA in E. coli Type Relative amount Mass # of Nucleotides ____________________%_________________kDa_____________________ 1.2X103 0.6X103 3.6X101 3700 1700 120 15 2.5X101 75 mRNA 5 heterogeneous sn RNA <1 rRNA 80 tRNA 23S 16S 5S RNA synthesis • RNA synthesis involves transcribing a specific portion of DNA strand into RNA sequence •RNA polymerases sequentially add ribonucleotides to the 3’ end of an RNA polymer using DNA strand as a template (5’ 3’ direction) 2Pi (RNA)n bases + NTP (RNA)n+1 bases +PPi •RNA Pol selects ribonucleotides complementary to the DNA template and catalyzes the formation of new phosphodiester bonds. This process is repeated as the enzyme moves along the DNA. RNA Synthesis: addition of new NTPs follows Watson-Crick rules N O N H 2N NH N N N NH 2 O N N N NH 2 N N O O A•U G•C Template base G C U A HN Incoming base C G A U The sequence of the RNA transcript is complementary to the transcribed DNA strand and is the same as the coding strand Coding 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’ Non-coding (template) strand Transcription 5’- A U G G C C U G G A C U U C A…3’ Translation …Met-Ala-Trp-Thr-Ser… Peptide