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Molecular Biology ( 1) part 2

Molecular Biology ( 1) part 2. Mamoun Ahram, PhD Second semester, 2015-2016. DNA replication a general mechanism. Resources. This lecture Campbell and Farrell's Biochemistry, Chapter 10. Transfer of molecular information. Some basic information.

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Molecular Biology ( 1) part 2

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  1. Molecular Biology (1)part 2 Mamoun Ahram, PhD Second semester, 2015-2016

  2. DNA replicationa general mechanism

  3. Resources • This lecture • Campbell and Farrell's Biochemistry, Chapter 10

  4. Transfer of molecular information

  5. Some basic information • The entire DNA content of the cell is known as genome • DNA is organized into chromosomes • Bacterial genome: usually one and circular chromosome • Eukaryotic genome: multiple, linear chromosomes complexed with proteins known as histones

  7. Bidirectionally…speaking • Replication moves progressively along the parental DNA double helix bidirectionally. • Because of its Y-shaped structure, this active region is called a replication fork.

  8. New DNA (long vs short) • A long strand and shorter pieces (Okazaki fragments) of DNA are present at the growing replication fork

  9. Components of DNA replication

  10. RNA primer • In order for the DNA polymerase to initiate replication, it requires a RNA primer, to be added first complementary to the DNA template • It is synthesized by a primase

  11. DNA helicases and SSB proteins • For DNA synthesis to proceed, the DNA double helix must be opened up ahead of the replication fork • Opening up the DNA is done by two types of protein contribute to this process • DNA helicases and • single-strand DNA-binding proteins

  12. DNA helicases • DNA helicases use ATP to open up the double helical DNA as they move along the strands. • Helicases form a complex with the primase called primosome.

  13. Single-strand DNA-binding (SSB) proteins • Single-strand DNA-binding (SSB) proteins bind tightly to exposed single-stranded DNA strands without covering the bases, which therefore remain available for templating • These proteins: • prevent the formation of the short hairpin structures • protect single-stranded DNA from being degraded • aid helicases by stabilizing the unwound, single-stranded conformation

  14. DNA polymerases in prokaryotes • DNA polymerase III: DNA polymerization at the growing fork in E. coli • The complex of primosome and polymerase is known as replisome • DNA polymerase I: • 5‘-to-3' exonuclease activity (removal of RNA primer) of each Okazaki fragment. • Fills in the gaps between the lagging-strand fragments. • DNA repair • DNA polymerase II, IV, and V : DNA repair

  15. DNA polymerase III • The DNA polymerase III is a very large protein composed of 10 different polypeptides • The core polymerase is composed of three subunits: • α subunit contains the active site for nucleotide addition • subunit is a 3-to-5 exonuclease that removes incorrectly added (mispaired) nucleotides from the end of the growing chain •  subunit stimulate the exonuclease activity

  16.  subunit • The  subunit, forms a clamp around DNA and holds the catalytic core polymerase near the 3 terminus of the growing strand

  17. How accurate is DNA replication? • The frequency of errors during replication is only one incorrect base per 109 to 1010 nucleotides incorporated • Why is fidelity high? • Hydrogen base-pairing is highly stable between G and C and between A and T. So, the DNA polymerase can catalyze the formation of phosphodiester bonds when the right hydrogen bonding takes place between the correction bases. • Proofreading mechanism (a 3’5’ exonuclease activity)

  18. DNA topoisomerases • A swivel is formed in the DNA helix by proteins known as DNA topoisomerases • A DNA topoisomerase breaks then re-forms phosphodiester bonds in a DNA strand. • Topoisomerase I produces an transient single-strand break (or nick) • ATP-independent • Topoisomerase II is responsible for untangling chromosomes by making a transient double-strand break • also known as gyrase in bacteria • ATP-dependent

  19. Replication Origin in bacteria • Bacterial replication starts at a origin known as origin of replication (OriC) • oriC regions contain repetitive 9-bp and AT-rich 13-bp sequences • 9-mer: binding sites for the DnaA protein • 13-mers: AT-rich region • facilitates separation of the double strand DNA

  20. Possible mechanism • When DnaA protein binds to 9-mers, it applies stress on the AT-rich region resulting in DNA "melting“.

  21. Two replication forks • The two replication forks proceed in opposite directions until they meet up roughly halfway around the chromosome.

  22. Origins of replication in human genome • An average human chromosome may have several hundred replicators (origins of replication).

  23. DNA polymerase in eukaryotes • Eukaryotic cells contain 9 DNA polymerases; most of them for DNA repair.

  24. The mechanism of replication • DNA polymerase  is responsible for synthesis of leading strand. • The DNA primase is part of the DNA polymerase α. • Polymerase α begins each Okazaki fragment on the lagging strand with RNA before passing the DNA to DNA polymerase . • DNA polymerase  then synthesizes the remainder of each Okazaki fragment. • The polymerases do not have a 5’3’ exonuclease • The primer is removed by two special enzymes. • DNA polymerase then fills in the gap.

  25. Roles of other polymerases • Polymerase  may function primarily in the repair of DNA damage • , , , and  DNA polymerases • Replication and repair of damaged DNA • No 3' to 5' exonuclease activity

  26. Role of chromatin • It seems that the timing of replication is related to the packing of the DNA in chromatin • In eukaryotes, chromosome duplication requires that DNA is freed from hisotnes and that new chromosomal histones be assembled onto the DNA behind each replication fork

  27. Chromatin remodeling • As a replication fork advances, it must pass through the parental nucleosomes • The chromatin-remodeling proteins free DNA from histones in order for enzymes to move along the DNA • The addition of histones to the newly synthesized DNA is aided by chromatin assembly factors (CAFs), which are proteins that package the newly synthesized DNA

  28. Role of telomeres • As the growing fork approaches the end of a linear chromosome, the lagging-strand template is not completely replicated • When the final RNA primer is removed, there is no place onto which DNA polymerase can build to fill the resulting gap • This would lead the lagging-strand to be shortened at each cell division

  29. Primer gap

  30. Telomerase • The enzyme that prevents this progressive shortening of the lagging strand is a modified enzyme called telomerase, which can elongate the lagging-strand template

  31. The mechanism • Telomere DNA sequences consist of many repeats of a short sequence • In humans, this sequence is GGGTTA, extending for about 10,000 nucleotides. Telomerase recognizes the telomere DNA repeat sequence and elongates it in the 5-to-3 direction • The telomerase synthesizes a new copy of the repeat, using an RNA template/primer that is a component of the enzyme itself

  32. How do we age? • As we grow older, the activity of telomerase is reduced • An inverse relationship between age and telomeric length has been observed • The gradual shortening of the chromosome ends leads to cell death, and it has even been suggested that life span is determined by the length of telomeres

  33. Elixir of youth

  34. DNA mutations http://www.ncbi.nlm.nih.gov/books/NBK21897/

  35. Types of mutations • Micromutation that involve small regions of the DNA • Macromutations that involve the chromosomes as a whole

  36. DNA micromutations • Single-base mutations can result in different types of mutations such as: • missense: a change of an amino acid • nonsense: premature termination of protein synthesis • frameshift: altering the reading of amino acid sequence • silent: does not lead to any change at the protein level, but contributes to genetic variability among individuals

  37. DNA micromutations (cont.) • Translocations, that bring different regions of gene segments together • Deletions of a few nucleotides to long stretches of DNA, • Insertions and duplications of nucleotides or long stretches of DNA • Inversion of DNA segments

  38. Causes of DNA mutation • DNA mutations can arise spontaneously or induced. • Spontaneous mutations are naturally occurring mutations and arise in all cells. • They arise from a variety of sources, including errors in DNA replication and spontaneous lesions • Induced mutations are produced when an organism is exposed to a mutagenic agent, or mutagen.

  39. Errors in DNA replication • An error in DNA replication can occur when an inaccurate nucleotide pair (say, A-C instead of G-C) forms in DNA synthesis, leading to a base substitution • Each of the bases in DNA can appear in one of several forms, called tautomers.

  40. Properties of Pyrimidines & Purines Keto-enol tautomerism • Tautomers are constitutional isomers of organic compounds that readily interconvert by a chemical reaction.

  41. Following DNA replication, tautomers lead to either transition or transversion mutations.

  42. Transitions • A purine substitutes for a purine or a pyrimidine for a pyrimidine

  43. Transversions • In transversion mutations, a pyrimidine substitutes for a purine or vice versa • In this case, a replication error would require mispairing of a purine with a purine or a pyrimidine with a pyrimidine

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