DNA Repair Types Direct repair Excision repair Recombination 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.

AGT inhibitor O6-benzylguanine is in clinical trials to be used in conjunction with antitumor alkylnitrosoureas

AGT overexpression in tumors makes them resistant to alkylnitrosoureas

Combination therapy with O6-benzylguanine overcomes tumor resistance to alkylnitrosoureas

Excision Repair Takes advantage of the double-stranded (double information) nature of the DNA molecule. Four major steps: Recognize damage. Remove damage by excising part of one DNA strand. The resulting gap is filled using the intact strand as the template. Ligate the nick.

Antiparallel DNA Strands contain the same genetic information 5' 3' 5' 3' 5' 3' A :: T A :: T A :: T G ::: C G G ::: C Two polynucleotide chains running in the opposite directions Each base is H-bonded to a complementary base in the opposite strand 2. The two chains are held together by stacking (hydrophobic) interactions, hydrogen bonds, and electrostatic forces. 3. Base pairing is in the order A:T, which contains two hydrogen bonds, and G:C, which contains three hydrogen bonds. T :: A T :: A T :: A 3' 5' 3' 5' 3' 5' Original DNA duplex DNA duplex with one of the nucleotides removed Repaired DNA duplex

Base excision repair (BER) 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 O N N H N N O H X a n t h i e

Uracil DNA glycosylase removes deaminated C No Me group N H 2 O BER C N N H N O N O Cytosine Not normally present in DNA U r a c i l 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). 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: 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) G T

Genetic diseases associated with defective DNA repair Xeroderma Pigmentosum NER Hereditary nonpolyposis MMR colorectal 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 replication transcription translation Recall that the genetic information stored in DNA is retrieved in a two step process. Transcription generates a copy of RNA identical in sequence to one of the DNA strands, and translation converts the nucleotide sequence of RNA into aa sequence of the corresponding protein. DNA

RNA is a biopolymer consisting of ribonucleotide units 5’ RNA is usually single stranded but can form hairpin structures by folding over the same strand: G C U 5'-U•C•C•C•A•C A•U•U•U-3' G•C A•U C•G Loop Double- stranded region (stem) 3’

Structural differences between DNA and RNA

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

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) 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. 2Pi

RNA Synthesis: addition of new NTPs follows Watson-Crick rules NH 2 N H 2 N O N N NH N N HN N N N N NH 2 O O A•U G•C Template base Incoming base G C C G U A A U The addition of nucleotides follows Watson-Crick base pairing rules (G:C, A:T) by forming specific hydrogen bonds between nucleobases in the template strand and the incoming dNTPs. The likelyhood of phosphodiester bond formation is very low unless WC bonds are formed

Non-coding (template) strand 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