Gene Expression- Protein Synthesis Protein synthesis is the key to expression of biological information - Structural Protein: bones, cartilage - Contractile Proteins: myosin, actin - Enzymes - Transport Proteins - Regulatory Proteins: hormone, insulin - Protective Protein: Immunoglobulin - Storage Protein: egg
DNA replication Eukaryotic Prokaryotic
DNA replication
DNA replication DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. This process is "replication" in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule. Cellular proofreading and error toe-checking mechanisms ensure near perfect fidelity for DNA replication. [1][2] In a cell, DNA replication begins at specific locations in the genome, called "origins". [3] Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA.
Semi-conservative replication, in which the daughter molecules each contain one polynucleotide derived from the original molecule and one newly synthesized strand
DNA replication 1) Bidirectional replication 2) DNA polymerase 3) How DNA polymerase uses to be bidirectional replication 4) DNA topoisomerase 5) DNA polymerase application: PCR and sequencing
Bidirectional Replication John Carins experiment in 1960, replication of the E. coli chromosome
Using autoradiography: incorporating a radioactive label into a substance and then placing the radioactive substance in contact with photographic emulsion so that it can take a picture itself First generation has one labeled strand (blue). The second round replication, unlabeled parental strand will pick up labeled partner and become singly labeled(a). The labeled parental strand will obtain a labeled partner and become doubly labeled(b). This doubly labeled part should expose the film more and therefore appear darker than rest of the DNA (loop A and C) http://en.wikipedia.org/wiki/john_cairns_(biochemist)
Elizabeth Gyurasits and R.B. Wake showed clearly DNA replication in Bacillus subtilis is bidirectional. These investigators strategy was to allow B. subtilis cells to grow for a short time in the presence of a weakly radioactive DNA precursor, then for a short time with more strongly radioactive precursor. These short bursts of labeling with a radioactive substance are called pulses of label. (3H-thymidine. Tritium(3H). In the figure the thick labeled showed very strongly at near both forks in bubble. The replication rate is the same in both direction
DNA polymerase Synthesize new daughter strands of DNA An enzyme able to build up a new DNA polynucleotide using an existing DNA strand as a template called a DNA dependent DNA polymerase DNA polymerase synthesize DNA but can also degrade it: The sequence of the new polynucleotide is dependent on the sequence of the template. DNA polymerization can occur only in the 5 3 direction
5-3 direction of DNA polymerase Template-dependent synthesis DNA DNA polymerase can add free nucleotides only to the 3' end of the newly forming strand. This results in elongation of the newly forming strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide only on to a pre-existing 3'-OH group DNA polymerase cannot initiate DNA synthesis unless there is already a short double stranded region to act as a primer
DNA polymerase can degrade polynucleotides as well as synthesize them 3 5 exonuclease, 5 3 exonuclease Enable a template-dependent DNA polymerase to remove nucleotides from the 3 end of a strand that it has just synthesized: Proofreading
Some polymerases are capable of both activities, while others only one or neither of them DNA polymerase I: 1957 DNA polymerase II and III: later found: 1972 Mutated in DNA pol I still keep replication function II and III found DNA polymerase II: mainly proofreading
Replication origin The replisome assembles at the origin, Replication fork
Replication Requires a Helicase and a Single-Strand Binding Protein Replication requires a helicase to separate the strands of DNA using energy provided by hydrolysis of ATP. A single-stranded binding protein is required to maintain the separated strands.
DnaA-ATP binds to short repeated sequences and forms an oligomeric complex that melts DNA. A hexamer of DnaB forms the replication fork. Helicase and SSB are also required. A short region of A-T-rich DNA is melted. DnaG is bound to the helicase complex and creates the replication forks. primase A type of RNA polymerase that synthesizes short segments of RNA that will be used as primers for DNA replication. SSBs attach to the unpaired polynucleotides produced by helicase action and prevent the strands from basepairing with one another or being degraded by nucleases
Initiation: Creating the Replication Forks at the Origin oric Six DnaC monomers bind each hexamer of DnaB, and this complex binds to the origin.(single strand binding protein) Prepriming involves formation of a complex by sequential association of proteins, which leads to the separation of
DNA Polymerases Are the Enzymes That Make DNA DNA is synthesized in both semiconservative replication and DNA repair reactions.
The DNA polymerase is dependent on the sequence of the template and is determined by complementary base pairing and DNA polymerization can occur only in the 5 to 3 direction
DNA polymerases are a family of enzymes that carry out all forms of DNA replication. [5] A DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. To begin synthesis of a new strand, a short fragment of DNA or RNA, called a primer, must be created and paired with the template strand before DNA polymerase can synthesize new DNA. The primase-helicase complex is used to unwind dsdna and synthesizes the lagging strand using RNA primers [4] The majority of primers synthesized by primase are two to three nucleotides long. [4]
Once a primer pairs with DNA to be replicated, DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds DNA polymerases are generally extremely accurate, making less than one error for every 10 7 nucleotides added. Even so, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a strand in order to correct mismatched bases. If the 5' nucleotide needs to be removed during proofreading, the triphosphate end is lost. Hence, the energy source that usually provides energy to add a new nucleotide is also lost
DNA polymerase I: Repair synthesis replaces a short stretch of one strand of DNA containing a damaged base DNA is synthesized by adding nucleotides to the 3 OH end of the growing chain, so that the new chain grows in the 5 to 3 direction
Bidirectional Replication?
Okazaki fragments On the left you see that at very short times of labeling (short pulses) very short pieces of DNA are found (2 sec, 7 sec, 15 sec). However, with longer and longer times, the pieces of DNA get increasing longer (120 sec). He then tried the same experiment with a mutant virus that was defective in a gene called DNA ligase. We will see that this is the enzyme that joins pieces of DNA together into larger structures. In this case (on the right) the labeled pieces of DNA remained short, even after long times of radiolabeling. Okazaki discovered the way in which the lagging strand of DNA is replicated via fragments by conducting an experiment using E. coli. After reacting E. coli with 3[H] Thymidine to synthesize DNA for only ten seconds, he placed the sample in a test tube of alkaline sucrose. The larger, heavier DNA flowed to the bottom of the test tube, while the smaller lighter DNA did not. When samples were taken from the bottom of the test tube, it was found that half were heavy and half were light, proving that half of the DNA was complete and half was in fragments. Then he took a sample of E.coli DNA that had been synthesized for an additional five seconds, and found all the activiy now resulted in the larger molecular weight. Therefore, there were no longer any fragments. This proved that during the five second chase, the RNA primer was removed and the bases were joined together by DNA polymerase I, leaving the new DNA fully mature and repaired.
RNA primers?
Priming of DNA synthesis Replication M13 phage DNA DNA polymerase is involved in M13 phage replication However, rifampicin (inhibit RNA polymerase) inhibit replication of M13 phage Since DNA polymerase is incapable of initiating DNA synthesis by itself, it needs a primer to supply a free 3 -end upon which it can build the nascent DNA. Very short pieces of RNA serve this priming function The DNA-degrading enzyme DNase cannot completely destroyed Okazaki and his wife to obtained intact primer from in lacked ribonuclease H or nuclease activity of DNA polymerase I. To label only intact primer, they used a capping enzyme that added GMP to the end of RNAs
Prokaryotic DNA polymerases Pol I: implicated in DNA repair; has 5'->3' (Polymerase) activity and both 3'->5' exonuclease (Proofreading) and 5'->3' exonuclease activity (RNA Primer removal). Pol II: involved in reparation of damaged DNA; has 3'->5' exonuclease activity. Pol III: the main polymerase in bacteria (elongates in DNA replication); has 3'->5' exonuclease proofreading ability.
Arthur Kornberg (March 3, 1918 October 26, 2007) was an American biochemist who won the Nobel Prize in Physiology or Medicine 1959 for his discovery of "the mechanisms in the biological synthesis of deoxyribonucleic acid (DNA)" together with Dr. Severo Ochoa of New York University Roger Kornberg was awarded the Nobel Prize in Chemistry in 2006 for his studies of the process by which genetic information from DNA is copied to RNA, "the molecular basis of eukaryotic transcription. RNA polymerase II
Polymerase I (most of replication in E. Coli) Pol I possesses three enzymatic activities: A 5' -> 3' (forward) DNA polymerase activity, requiring a 3' primer site and a template strand A 3' -> 5' (reverse) exonuclease activity that mediates proofreading A 5' -> 3' (forward) exonuclease activity mediating nick translation during DNA repair. (fill Okazaki fragment)
DNA Polymerases Have Various Nuclease Activities DNA polymerase I has a unique 5 3 exonuclease activity that can be combined with DNA synthesis to perform nick translation.
DNA Polymerases Control the Fidelity of Replication High-fidelity DNA polymerases involved in replication have a precisely constrained active site that favors binding of Watson Crick base pairs. processivity The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle.
DNA Polymerases Control the Fidelity of Replication DNA polymerases often have a 3 5 exonuclease activity that is used to excise incorrectly paired bases. The fidelity of replication is improved by proofreading
DNA Polymerases Have a Common Structure Many DNA polymerases have a large cleft composed of three domains that resemble a hand. DNA lies across the palm in a groove created by the fingers and thumb. Structure from Protein Data Bank 1KFD. L. S. Beese, J. M. Friedman, and T. A. Steitz, Biochemistry 32 (1993): 14095-14101.
The Two New DNA Strands Have Different Modes of Synthesis The DNA polymerase advances continuously when it synthesizes the leading strand (5 3 ), but synthesizes the lagging strand by making short fragments (Okasaki fragments) that are subsequently joined together. semidiscontinuous replication The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously.
The Two New DNA Strands Have Different Modes of Synthesis 5 3 3 5
Okazaki Fragments Are Linked by Ligase Each Okazaki fragment starts with a primer and stops before the next fragment. DNA polymerase I removes the primer and replaces it with DNA.
Okazaki Fragments Are Linked by Ligase DNA ligase makes the bond that connects the 3 end of one Okazaki fragment to the 5 beginning of the next fragment. Ligase mutated E.Coli.
DNA polymerase III can synthesize DNA only for a certain distance before it reaches the RNA primer at the 5 end of the next Okazaki fragment. DNA polymerase III stops and DNA polymerase I come into action, continuing DNA
Replication of the E.coli genome terminates within a defined region Recognition site called Tus a sequencespecific DNA-binding protein. The six
Topoisomerases release the strain in replicating circular DNAs Cairns recognized a swivel in the DNA duplex that would allow the DNA strands on either side to rotate to relieve the strain Topoisomerase I and II single strand break and both strand break and reseal.