DNA Topoisomerases relieve the supercoiling stress ahead of the fork

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Peregrine Robertson
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1 DNA Topoisomerases relieve the supercoiling stress ahead of the fork

2 Tw 1) T w : # of turns around the central axis 2) W r : # of times the double helix crosses itself 3) Linking Number: L k = T w + W r Wr

3 L k = T w + W r The linking number of a closed circular molecule cannot be changed without breaking bonds (Tw and Wr can interconvert)!

4 The enzymes which can alter DNA supercoiling are called topoisomerases, and they come in two varieties: Type I: cut one strand Changes Lk in steps of 1 Enzyme nicks one strand and passes the other strand Example: Topo I Type II: cut two strands Changes Lk in steps of 2 Enzyme breaks both strands and passes another duplex through the break use ATP Example: DNA gyrase BOTH types form a covalent protein-dna intermediate through key tyrosine residues of the enzymes.

5 DNA Gyrase Targeted by ciprofloxacin and nalidixic acid

6 Number of base pairs = 400 For B-DNA, assume 1 turn = 10bp Calculate the Twist, Writhe and Linking Number of this relaxed DNA molecule. Twist = 400/10 = 40 Writhe = 0 Lk = Tw+ Wr = = 40

Molecule A Twist = 400/10 = 40 Writhe = 0 Lk = Tw+ Wr = 40 + 0 = 40 DNA Gyrase Introduces four right-handed supercoils Molecule

7 Molecule A Twist = 400/10 = 40 Writhe = 0 Lk = Tw+ Wr = = 40 DNA Gyrase Introduces four right-handed supercoils Molecule B # of base pairs = 400 Molecule C No enzyme action (hint no bonds broken!) Just unwind Molecule B by removing the 4 supercoils

8 Prokaryotes vs Eukaryotes Circular vs. Linear One origin of replication vs. Many origins of replication Several DNA polymerases (with mechanisms of action analogous to bacterial DNA polymerases)

9 NOT A HAPPY ENDING THE END-REPLICATION PROBLEM! Primer removal Replication leading strand lagging strand Ligate Okazaki fragments

10 Telomerase extends 3 ends of chromosomes Additional 3 end DNA acts as a template for a new Okazaki fragment Maturation of Okazaki fragment primer gap Telomere repeats New Okazaki fragment

TELOMERASE BRINGS ITS OWN TEMPLATE 5 3 Telomerase with its own RNA template (5 CCCUAACCC3 ) 5 3 5 3 5 3 5 3 Elizabeth Blackburn, Carol Greider,

11 TELOMERASE BRINGS ITS OWN TEMPLATE 5 3 Telomerase with its own RNA template (5 CCCUAACCC3 ) Elizabeth Blackburn, Carol Greider, Jack Szostac shared the 2009 Noble Prize in Physiology/Medicine for their ground-breaking discoveries of telomeric repeat sequences and telomerases.

12 DNA Repair A fundamental difference from RNA, protein, lipid, etc. All these others can be replaced, but DNA must be preserved! All repair mechanisms follow similar path: detection, removal, repair

13 DNA Repair after Replication If mismatches escape proofreading, mismatch repair occurs after DNA synthesis is complete DNA Polymerase and Proofreading ~10-7 mis-match repair after replication ~10-2 combined error rate ~10-9!

15 How does the E.coli mismatch repair system know which of the two mismatched nucleotides to replace? G or T?

16 Newly synthesized DNA is hemi-methylated CH 3 CH 3 old strand (methylated by dam methylase) New strand (no methyls) CH 3 CH 3 CH 3

17 Human nonpolyposis colorectal cancer is associated with defects in a human counterpart to 1) MutS (= hmsh2) or 2) MutL (=hmlh1) 1/200 Americans is affected (15% of all colon cancers) HNPCC patients have 80% lifetime risk of colon cancer

18 What else causes DNA damage? Exogenous DNA damage UV rays in sunlight environmental chemicals Endogenous DNA damage oxidative damage deamination

20 Diverse DNA repair systems Mostly characterized in bacteria General mechanisms shared in eukaryotes 1. Mismatch excision repair 2. Direct repair e.g. photolyase 3. Base excision repair 4. Nucleotide excision repair 5. Double-strand break repair and recombination

21 Base Excision Repair (BER)

22 Nucleotide Excision Repair (NER) xeroderma pigmentosa mutations in XP genes (A-G) XP proteins involved in NER in humans

23 Don t need to know this list, but just realize that several human diseases are linked to impaired DNA repair systems.

24 Ames Test

25 Transcription Unlike DNA replication, we want to copy information only from specific sequences ( genes ) at specific times in specific cell types Need specific start and stop signals Although the DNA template of each gene is double stranded, only one strand is transcribed

26 RNA Polymerase: polymerizes in the 5 to 3 direction Base pairing of hybrid (DNA/RNA) follows Watson- Crick pairing rules dictated by DNA template strand Primer is not required, but does need ds DNA template error rate ~ 1 in 10 5

27 E. coli RNA polymerase only one RNA polymerase transcribes all types of RNA mrna, trna and rrna Holoenzyme α - 2 copies (small 37kD protein) β - 1 copy (large 151kD protein) β - 1 copy (large 155kD protein) σ - 1 copy Sigma subunit is required for initiation of transcription. Is released once RNA chain is a few bases long (elongation phase) Core Beta subunit has the RNA polymerse activity. The antibiotic Rifampicin binds here and inhibits initiation of transcription.

28 Steps of transcription 1. Initiation 2. Elongation 3. Termination

29 How does RNA Pol know where to start (initiate) transcription?

30 RNA Polymerase

31 How to define a consensus? Compare many DNA sequences bound by RNA Polymerase to identify a consensus -1 base +1 base For theses 5 sequences: T 80 A 80 T 80 G/C 40 T 40 T 80

32 The prokaryotic consensus sequence T 78 T 82 G 68 A 58 C 52 A T 82 A 89 T 52 A 59 A 49 T 89 Or more simply: Spacer Size 17bp spacer Pribnow box

33 17bp spacer In general, the more similar the sequence is to the consensus, the more efficient the promoter & vice versa More efficient promoter = stronger promoter = more transcription of gene Less efficient promoter = weaker promoter = less transcription of gene

34 17bp spacer Molecular weight of sigma factor This is the consensus sequence recognized by σ 70 Enables RNA Pol to specifically bind to promoter sequences Required for efficient initiation Is release after RNA chain is a few base pairs long There are multiple forms of σ (sigma) Different sigma factors allow differential gene expression Each sigma factor recognizes a unique promoter

35 Sigma Factor -35 Spacer -10 class of genes σ 70 TTGACA 17 TATAAT Housekeeping σ 38 CTATACT Stationary phase σ 32 CTTGAA CCCATNT Heat shock/stress σ 54 CTGGGNA 5-12 TTGCA Nitrogen metabolism

37 Closed Promoter complex Open promoter complex

38 DNA Template T (or C) Base 2 Notice the 2 -OH This is RNA!!! RNA Product

40 During Elongation Sigma factor dissociates and can recycle Rate of elongation: ~50 nt/sec DNA rewinds behind the transcription bubble

42 After the RNA polymerase moves into elongation phase and downstream from the promoter (called promoter clearance), another molecule of RNAP can bind to the promoter

43 Electron micrograph of a DNA sequence that is being actively transcribed Miller spread or Christmas tree spread Direction of transcription Image from: French, S.L. et al, MOLECULAR AND CELLULAR BIOLOGY, 2003, Vol. 23, No. 5 p

44 Termination Rho independent Rho dependent

45 GC-rich hairpins poly(u) stretch Rho(Protein)-Independent Termination

46 Rho-Independent Termination Termination signal contained within the transcribed region of the DNA template Note that while promoter was not transcribed into RNA, the terminator sequence is transcribed

48 Rifampicin Binds specifically to bacterial RNA polymerase Inhibits INITIATION of transcription (does not hinder RNA chain elongation)

49 Actinomycin D Intercalates into DNA double helix and is also a groove binder Can inhibit both prokaryotic and eukaryotic transcription Used in the treatment of some cancers

Structure of a gene Promoter sequences +1 5 3 ATGTACTGAT

50 Structure of a gene Promoter sequences ATGTACTGAT TACATGACTA 3 5 The RNA transcribed from this gene will start with a)acugau

51 RNA Modification and Processing In prokaryotes mrna is not processed Transcription and translation can be coupled

52 In contrast to the prokaryotic mrna, the prokaryotic trna and rrna are significantly modified and processed! primary transcript

55 Structure of a gene gene protein 5 UTR = 5 untranslated region 3 UTR = 3 untranslated region When the sequence of a gene is provided, by convention it is the sequence of the non-template strand starting from the +1 of transcription and written in the 5 to 3 direction.

DNA Transcription. Dr Aliwaini

DNA Transcription. Dr Aliwaini DNA Transcription 1 DNA Transcription-Introduction The synthesis of an RNA molecule from DNA is called Transcription. All eukaryotic cells have five major classes of RNA: ribosomal RNA (rrna), messenger