DNA Replication DNA by Watson Crick Franklin When
DNA Replication
DNA by Watson & Crick & Franklin When discovered, the structure suggested how DNA was able to replicate: n The H-bonds between complementary bases break n This n allows the DNA to unzip Each DNA strand then acts as a template to build the complimentary strand n This results in two identical DNA molecules; one for each daughter cell
The big question one time was the method of DNA replication; was it: 1) Conservative: the original ds. DNA was conserved as one molecule for one daughter, and a completely new one for the other daughter, or 2) Semi-conservative: the original molecule is split in half, and the other side is filledin) 3) Dispersive: Each new molecule is comprised of bits and pieces of both new DNA and the original strand
Can you design a nifty experiment to verify? Models of DNA Replication n Alternative models n become experimental predictions conservative P 1 2 semiconservative dispersive
Meselson and Stahl n grew Escherichia coli (E. coli. ) in 15 N media, which made the DNA heavy. n Then grew it on normal media (14 N). n 2 outcomes: 1)New molecules have medium (hybrid) DNA or 2) One molecule has heavy DNA the other light, n The first was observed
That process is called Semiconservative Replication
Overview. n DNA replication has many steps, but in general: Initiation and unzipping. n Priming n Elongation n Proof-reading. n
Initiation and Unzipping n Replication begins when proteins bind at a specific site on the DNA known as the origin of replication (ori). n n Eukaryotic replication is similar to prokaryotic replication but more complex The closed circular DNA of prokaryotes usually only has one origin of replication (ori) n Linear eukaryotic DNA has multiple ori’s
DNA Strand Separation n The strands cannot simply be pulled apart because they are held together by hydrogen bonds and twisted around each other in the double helix. Specific enzymes work together to expose the DNA strands n
DNA Strand Separation n What is involved? n DNA helicase: unwinds the double helix by breaking the H-bonds at the replication fork. Replication fork: region where enzymes replicating DNA bind to an untwisted, s. s. DNA strand.
DNA Strand Separation n Single-stranded Binding Proteins (SSBs): bind the exposed DNA template strands to block new Hbonds that would re-join the strands
DNA Strand Separation n DNA gyrase: relieves tension from the unwinding of the DNA strands. It cuts nicks in both strands of DNA, allowing them to swivel around one another and then resealing the cut strands.
Replication begins in 2 directions from the ori ’s as a region of the DNA is unwound. n DNA replication proceeds toward the direction of the replication fork (leading strand) on one strand away from the fork (lagging strand) on the other strand. n In eukaryotes, when 2 replication forks are too near, a replication bubble forms n
Building Complimentary Strands n In prokaryotes, there are 3 enzymes known to function in replication & repair n DNA polymerase I, II & III In eukaryotes, there are 5 enzymes known to function in replication & repair n
Priming n n Once the replication bubble is started, then Primase lays-down short-sequence RNA primers as complements to the DNA strand. The DNA replication complex (lots of enzymes) sits on the primed site and starts to build on the RNA primer.
RNA primers are synthesized by primase and are temporary n The leading strand (uses 3’-5’ template) is synthesized continuously n The lagging strand (uses 5’-3’ template) is synthesized discontinuously in short fragments n
Elongation DNA polymerase III builds the complimentary strand of DNA n DNA polymerase III adds complimentary nucleotides (deoxyribonucleoside triphosphates) in the 5’ to 3’ direction, using RNA primers as starting points n The RNA primers are needed because DNA Pol III needs a 3’OH to add to. n The lagging strands produces many small segments called Okazaki fragments n
Energy of Replication Where does energy for bonding usually come from? We come with our own energy! You remember ATP! Are there other ways energy to get energy nucleotides? out it? You of bet! energy ATP CTP TTP GTP modified nucleotide And we leave behind a nucleotide! CMP TMP GMP ADP
n Energy of Replication The nucleotides arrive as nucleosides n DNA bases with P–P–P n P-P-P = energy for bonding DNA bases arrive with their own energy source for bonding n bonded by enzyme: DNA polymerase III n ATP GTP TTP CTP
Replication n Adding bases n can only add nucleotides to 3 end of a growing DNA strand n need a “starter” nucleotide to bond to n strand only grows 5 3 B. Y. O. ENERGY! The energy rules the process 5 3 energy DNA Polymerase III 3 5
n DNA polymerase I removes the RNA primers from the leading and the lagging strand replaces them with the appropriate deoxyribonucleotides. Text page 221, figure 7 b
n DNA ligase joins the Okazaki fragments into one strand on the lagging strand of DNA through the formation of a phosphodiester bond.
As the 2 new strands of DNA are synthesized, 2 d. s. DNA molecules are produced that automatically twist into a helix.
Editing & proofreading DNA n 1000 bases/second = lots of typos! n DNA polymerase I n proofreads & corrects typos n repairs mismatched bases n removes abnormal bases n repairs damage throughout life n reduces error rate from 1 in 10, 000 to 1 in 100 million bases
Telomeres n n Since RNA primers are needed, they can never be placed at the EXACT end of the chromosome, so some DNA will be lost (chromosomes shorten) at every replication. Thus, eukaryotic chromosomes have ‘caps’ on their two ends of a highly repetitive sequence, called telomeres. When these are gone, then coding DNA (genes) are lost. This has been suggested as a reason for aging. Immortal cells (stem cells, zygotes, germ-line cells) have an active telomerase enzyme that adds back to the telomeres.
Chromosome erosion All DNA polymerases can only add to 3 end of an existing DNA strand Houston, we have a problem! DNA polymerase I 5 3 3 5 5 growing replication fork 3 DNA polymerase III RNA Loss of bases at 5 ends in every replication u chromosomes get shorter with each replication 5 3
Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap u limit to ~50 cell divisions 5 3 3 5 5 growing replication fork 3 telomerase 5 Telomerase u u u enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells § high in stem cells & cancers -- Why? TTAAGGGTTAAGGG 3
Fast & accurate! n It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome n n divide to form 2 identical daughter cells Human cell copies 6 billion bases & divide into daughter cells in only few hours remarkably accurate n only ~1 error per 100 million bases n ~30 errors per cell cycle n
What does it really look like? 1 2 3 4
What Does It Really Look Like, (3 D Animated Movie Version)
The “Central Dogma” n Flow of genetic information in a cell transcription DNA replication translation RNA protein
Review Questions
Base your answers to the following questions on the choices below: A. Helicase B. Polymerase C. Ligase D. Primase 1. 2. 3. Brings together the Okazaki fragments. Adds nucleotides to the leading strand. Recognizes the origin of replication in the DNA.
4. In DNA synthesis, DNA is A. B. C. D. Read 3’ to 5’ and made 5’ to 3’ Read 3’ to 5’ and made 3’ to 5’ Read 5’ to 3’ and made 5’ to 3;
4. A space probe returns with a culture of a microorganism found on a distant planet. Analysis shows that it is a carbon-based life form that has DNA. You grow the cells in 15 N medium for several generations and then transfer it to 14 N medium. Which pattern in this figure would you expect if the DNA were replicated in a conservative manner? a. b. c. d. e.
6. Imagine the following experiment is done: Bacteria are first grown for several generations in a medium containing the lighter isotope of nitrogen, 14 N, then switched into a medium containing 15 N. The rest of the experiment is identical to the Meselson and Stahl experiment. Which of the following represents the band positions you would expect after two generations? *
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