10-26-11 Part B DNA Replication, Repair and Recombination

1. 10-26-11 Part B DNA Replication, Repair and Recombination

2. Any successful information-based system involves conservation and transfer • DNA is the relatively stable structure that maximizes information storage and duplication • RNA is more reactive than DNA with numerous roles in protein synthesis and gene expression • Decoding DNA requires DNA-protein interactions • Major and minor grooves facilitate sequence specific binding • Numerous contacts are involved including: Hydrophobic interactions, hydrogen bonding and ionic bonds

3. Three-dimensional structures of DNA-binding proteins have surprisingly similar structures • Most possess a twofold axis of symmetry and can be separated into families: • 1. Helix-turn-helix • 2. Helix-loop-helix • 3. Leucine zipper • 4. Zinc finger • For example, many leucine zipper transcription factors form dimers as their leucine-containing a-helices associate via van der Waals forces

4. Zinc finger Helix-turn-helix Helix-loop-helix Leucine zipper

5. All living organisms most possess rapid and accurate DNA synthesis and effective DNA repair mechanisms • Variation may also be important for adaptability to environments • Variation is caused by genetic recombination and mutation • Prokaryotes such as E. coli have been excellent subjects for investigation of these mechanisms, due to simple genetics and short generation time

6. DNA Replication • DNA replication must occur before cell division; the mechanism is similar in all living organisms: • After two strands have separated, each serves as a template for synthesis of a complementary strand • This process is referred to as semiconservative replication

7. Most DNA replication takes place at replication factories, which are relatively stationary during the process • DNA Synthesis in Prokaryotes - DNA replication in E. coli is a relatively complex process that consists of several basic steps: • 1) DNA unwinding requires helicases which are ATP-dependent enzymes that catalyze the unwinding of duplex DNA (e.g., DnaB in E. coli) • 2) Primer synthesis is the formation of short RNA segments (primers) required for the initiation of DNA replication by primase (e.g., dnaG) • 3) DNA synthesis is the synthesis of complementary DNA in a 5′3′ direction catalyzed by a large multienzyme complex referred to as DNA polymerase

8. DNA helicase separates DNA strands

9. Werner Syndrome is caused by defective helicase activity

10. Separation of DNA strands (unwinding) creates overwound DNA

11. Joining DNA fragments - frequently during DNA synthesis, DNA segments must be joined together • DNA ligase catalyzes the formation of the phosphodiester bond between adjoining nucleotides • Supercoiling control is accomplished by topoisomerases, which relieve torque in the DNA so the replication process is not slowed • Type I topoisomerases produce transient single-strand breaks, • cause spontaneous relaxation of supercoils. • Type II topoisomerases produce transient double-strand breaks, require ATP to add negative supercoils. • Bacterial Type II topoisomerase is called gyrase.

12. The DNA ligase reaction

13. Naladixic acid and Ciprofloxacin inhibit DNA gyrase, interfere with breakage and rejoining of DNA chains Novobiocin blocks ATP binding to gyrase

14. The DNA replicating machine (replisome) consists of two pol III holoenzymes, the primosome and DNA unwinding proteins • There are four other DNA polymerases: • DNA polymerase I is involved in RNA primer removal and replacement with DNA • DNA polymerase II, IV and V are involved in DNA repair • All three enzymes possess a proofreading 3′5′ exonuclease activity (e.g., the e subunit of pol III) • Pol I also has 5′3′ exonuclease activity

15. 3’ 5’Proofreading by DNA Polymerase

16. DNA proofreading by DNA polymerase necessitates synthesis of RNA primers to initiate DNA synthesis

18. Pol III holoenzyme is composed of at least 10 subunits (functions defined by mutations in E. coli). The core polymerase is formed of three subunits a, e, and  The b-protein (sliding clamp) is two subunits and forms a donut-shaped ring around the template DNA

21. In E. coli when the ATP/ADP ratio is high and there is enough DnaA, replication can begin at the initiation site (oriC) • Replication proceeds in both directions with each replication fork having helicases and a replisome • E. coli only has one origin of replication, making it a single replication unit (replicon) organism

22. Replication begins when DnaA proteins bind to multiple 9-bp sites (yellow) within the oriC The oligomerization of DnaA results in a nucleosome-like structure requiring ATP and histone-like protein (HU) Causes three 13 bp repeats near the DnaA-DNA complex to open

23. Replication Fork DnaB (a helicase), complexed with DnaC (a helicase loader) enters the open oriC region; once DnaB is loaded, DnaC is released The replication fork moves forward as DnaB unwinds the helix Topoisomerases relieve torque ahead of the replisome Single strands are kept apart by numerous copies of single- stranded DNA-binding protein (SSB)

24. DNA Replication at a Replication Fork • DNA synthesis only occurs in the 5′3′ direction, so one strand is continuously synthesized (leading strand) while the other is not (lagging strand) • The lagging strand is synthesized in short 5′3′ segments called Okazaki fragments (1,000–2,000 nucleotides)

25. E. coli DNA Replication Model • For pol III to initiate DNA synthesis an RNA primer must be present • On the leading strand, only a single primer is required • On the lagging strand, a primer is required for each Okazaki fragment

26. Pol III synthesizes at the 3′ end of the primer • RNA primers are removed by pol I which then synthesizes complementary DNA • DNA ligase then joins Okazaki fragments • Tandem operation of two pol III complexes requires the lagging strand be looped around the replisome

27. Despite the complexity and rate (1,000 base pairs per second per replication fork) of DNA replication in E. coli,it is amazingly accurate - one error per 109 or 1010 base pairs • This is due to the precise nature of the copying process (complementary), proofreading mechanism of DNA pol I and III, and postreplication repair mechanisms

28. DNA Synthesis in Eukaryotes has much in common with prokaryotes; they also have significant differences • Timing of replication - eukaryotic replication is limited to a specific phase of the cell cycle (S phase) • Replication rate is slower in eukaryotes (50 bp per second per replication fork) due to complex chromatin structure The Eukaryotic Cell Cycle

29. Replicons - eukaryotes have multiple replicons (about every 40 kb) to compress the replication of their large genomes into short periods • Humans have 30,000 origins of replication • Okazaki fragments are from 100 to 200 nucleotides long Multiple-Replicon Model of Eukaryotic Chromosomal DNA Replication

30. When the replication machinery reaches the 3′ end of the lagging strand, there is insufficient space for a new RNA primer • This leaves the end of the chromosome without its complementary base pairs • Chromosomes with 3′-ssDNA overhangs are very susceptible to nuclease digestion • Eukaryotes compensate with telomerase, a ribonucleoprotein with reverse transcriptase ability

32. The telomere consists of hundreds of tandem hexanucleotide repeats One strand is G rich at the 3’end and is slightly longer than the other The G rich strand may promote loop structures that protect the end of the The chromosome from 3’ exonucleases How are the repeated structures in telomeres generated? Elizabeth Blackburn and Carol Greider, working on telomere structure in Tetrahymena, discovered the enzyme responsible - telomerase

33. Telomerase has an RNA base sequence complementary to the TG-rich sequence of telomeres • Telomerase uses this sequence to synthesize a single-stranded DNA to extend the 3′ strand of the telomere • Afterward the normal replication machinery synthesizes a primer and Okazaki fragment

34. During normal human aging, the telomeres of somatic cells shorten over time • Once telomeres are reduced to a critical length, chromosome replication cannot occur • Telomere shortening causes cell death • 90% of all cancers have hyperactive telomerase • This fact makes telomerase a potential target for • anticancer drugs

35. DNA Repair • The natural rate of mutation is about 0.1 to 1.0 mutation per million gametes • Mutations can be small point mutations or large chromosomal abnormalities • Mutations are caused by the properties of the bases themselves, chemical processes or xenobiotics • Cells possess a great variety of DNA repair mechanisms

36. Guanine oxidation causes G to pair with A Caused by reaction with hydroxyl radical, A mutagen

37. Adenine deamination causes A to pair with C

38. Aflatoxin is activated by cytochrome P450 forming a reactive species that modifies bases

39. Most DNA repair mechanisms involve the removal of nucleotides • Mismatch repair corrects errors made during replication • Base excision repair is a mechanism that removes and then replaces individual nucleotides whose bases have undergone damage • A DNA glycosylase cleaves the N-glycosidic linkage between the damaged base and the deoxyribose • The resulting apurinic or apyrimidinic sites are resolved through the action of nucleases that remove the residue, DNA polymerase (pol I in bacteria; DNA polymerase b in mammals) and DNA ligase

40. Mismatch repair The parent strand is recognized by containing methylated adenine

41. Base excision repair corrects frequent conversions by deamination of 5-methylcytosine to thymine

42. Base excision repair

43. DNA contains thymine instead of uracil to permit repair of deaminated cytidine

44. Thymine dimers form in response to UV light, are removed by nucleotide excision repair

45. In nucleotide excision repair, bulky (2-30 nt) lesions are removed and the resulting gap filled • The excision enzymes of this process seem to recognize the distortion rather than the base sequence • In E. coli,the excision nuclease composed of Uvr A, B and C cuts the DNA and removes 12 to 13 nt ssDNA sequence containing the lesion

46. Recombination repair can eliminate certain types of DNA damage that are not eliminated before replication • Involves the exchange of a corresponding segment from a homologous chromosome

47. DNA Recombination • Recombination is the rearrangement of DNA sequences by exchanging segments from different molecules • Genetic recombination is a principle source of the variations that make evolution possible • Two types of recombination: • 1) General recombination occurs between homologous DNA molecules (most common during meiosis) • 2) Site-specific recombination - the exchange of sequences only requires short regions of DNA homology (e.g., transposition)

48. General Recombination requires the precise pairing of homologous DNA molecules • The currently accepted model was first proposed by Robin Holliday in 1964: • 1. Two Homologous DNA molecules become paired • 2. Two of the DNA strands, one in each molecule, are cleaved

49. 3. The two nicked strand segments cross over, forming a Holliday intermediate • 4. DNA ligase seals the cut ends • 5. Branch migration, caused by base-pairing exchange, leads to the transfer of a segment of DNA from one homologue to the other