Broad Course Objectives for DNA Replication Students will

Broad Course Objectives for DNA Replication Students will be able to: • describe the historic experiment that demonstrated DNA replication follows a semi-conservative model. • describe the process of DNA replication in prokaryotes at the biochemical level • explain how proofreading and repair is accomplished during DNA synthesis

Outline/study guide—DNA Replication • At what point in the cell cycle does DNA replication occur? • When two DNA molecules (or chromosomes) are made from one, where do the parental strands end up, vs. the newly synthesized strands? (i. e. semiconservative replication) Why can DNA only be synthesized in the 5’ 3’ direction? • • • What are the enzymes and proteins involved in DNA synthesis? What is the function of each and at what point do they act? At what point does RNA function in DNA replication? What determines the lagging strand vs. the leading strand? How does this change on the “other” side of the replication origin? How are the Okazaki fragments joined into one continuous DNA strand? • How does the DNA replication machinery correct errors made during replication? • • Are human chromosomes linear or circular? Bacteria? Why do linear chromosomes (but not circular chromosomes) have a problem with telomeres becoming shorter and shorter with each round of replication? How do some cells get around this?

48 year old woman with Werner Syndrome

Progeria

5′ Each strand of the parent DNA molecule becomes a template for the new molecule(s) 3′ 5′ C G A T C G T A GC GC T A A T C G A T GC GC T A C G A T C G Replication fork A T C 3′ G A G G T A The width of the nucleotides reflect larger purines and smaller pyrimidines 5′ T C C C T A Identical base sequences Lagging A T strand 5′ 3′ T A C G A T Incoming C G T A nucleotides T A GC C G T A G C A T A 5′ C G A T Original Newly Original GC (template) synthesized (template) G C strand daughter strand T A 3′ 5′ A T T A C G T A Leading strand G C C G T A 3′ (a) The mechanism of DNA replication Brooker Fig 13. 1 3′ 3′ 3′ C G A T C G T A GC GC T A A T C G A T GC GC T A 3′ 5′ (b) The products of replication Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

Different models of DNA Replication Original DNA First replication Second replication Conservative model Semiconservative model Dispersive model Brooker fig 13. 2 Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

How do you expect the different models to appear in the centrifuge experiment? Original DNA (N 15) First Replication (N 14) Second Replication (N 14) Conservative model Semiconservative model Dispersive model Brooker fig 13. 2 Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

Experiment to distinguish between DNA replication models Experimental level 1. Grow bacteria in excess of 15 N-containing compounds. Switch to 14 N at Generation 1. 15 N-DNA = purple 14 N-DNA = blue Conceptual level 14 N Generation 0 solution Add 14 N Suspension of bacterial cells labeled with 15 N 1 2. Incubate the cells for various cell generations 2 3. Lyse cells to release DNA 37°C Up to 4 generations DNA Cell wall Lysate 4. Load sample of lysate onto Cs. Cl gradient. 5. Centrifuge until the DNA molecules reach equilibrium densities. Cs. Cl gradient Cell membrane Density centrifugation Light DNA Half-heavy DNA 6. View DNA within the gradient using a UV light. Brooker fig 13. 3 Heavy DNA (after 2 generations. ) Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

Interpreting the Data Generations After 14 N Addition 4. 1 3. 0 2. 5 1. 9 1. 5 1. 1 1. 0 0. 7 0. 3 Light Half-heavy Heavy *Data from: Meselson, M. and Stahl, F. W. (1958) The Replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 44: 671− 682 After ~ two generations: DNA is “light” and “half-heavy” (Consistent with which model? ) After one generation, DNA is “half-heavy” (consistent with both semiconservative and dispersive models) Copyright ©The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display

Why does DNA (and RNA) only “grow” in the 3’ direction? New strand Template strand Sugar A Base Phosphate P T A T C G G C A T P 3’ end 5’ end P P OH P Pyrophosphate 3’ end C OH Nucleoside triphosphate (Like Brooker, fig 13 -15) C 2 P 5’ end Fig from Cambell and Reece, 7 th ed

Overview of DNA Replication Origin of replication DNA strands separate at origin Primers initiate DNA synthesis. Synthesis of the leading strand occurs in the same direction as movement of the replication fork. 1 st Okazaki fragment of lagging strand is made in opposite direction. Replication forks Leading strand 5′ 3′ 3′ 5′ Direction of replication fork Primer The leading strand elongates, and a second Okazaki fragment is made. Primer 3′ 5′ 1 st Okazaki fragment of the lagging strand 3′ 5′ 5′ 3′ 3′ 2 nd Okazaki fragment 1 st Okazaki fragment 5′ 3′ 5′ The leading strand continues to elongate. A third Okazaki fragment is made, and the first and second are connected together. 3′ 3′ 5′ 5′ 3 rd Okazaki fragment 3′ 5′ Brooker, fig 13. 10 First and second Okazaki fragments have been connected to each other. 3′ 5′ Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

Functions of key proteins involved with DNA replication • DNA helicase breaks the hydrogen bonds between the DNA strands. Origin Single-strand binding protein • Topoisomerase alleviates positive supercoiling. • Single-strand binding proteins keep the parental strands apart. DNA helicase 3′ 5′ DNA polymerase III Topoisomerase II • Primase synthesizes an RNA primer. • DNA polymerase III synthesizes a daughter strand of DNA. • DNA polymerase I excises the RNA primers and fills in with DNA (not shown). Leading strand RNA primer DNA polymerase III Replication fork 5′ Okazaki fragment Primase 3′ RNA primer Lagging strand Parental DNA • DNA ligase covalently links the Okazaki fragments together. DNA ligase 3′ 5′ Direction of fork movement Brooker, fig 13. 7 Copyright ©The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display Linked Okazaki fragments

E. coli chromosome Origin of Replication ori. C AT-rich region 5′ –GGA T CC T GGGT A T T AAAAAGAAGA T C T AT T T AGAGA T C T G T T C T AT CC T AGGACCC A T A A T T T C T AGA T AA AT AAA T CTCT AGAC AAGA T A 1 50 Dna. A box T GA T CT T AGGA T CGC A C T GCCCT GT GGA T AACA AGGA T CGGCT AC AC T AGAGA A TCCT AGCGT GACGGGACACCT A T TGT T CC T AGCCGA 51 100 Dna. A box T T T A AGA T CA A CCTGGA AAGGA T C AT T AA CTG T GAAT GA T CGG T GAT A A A T T C T AGT T GGACC T T T CC T AGT AA T T GAC ACT T AC T AGCC AC T A 101 150 Dna. A box CC T GGA CCGT A AGCTGGGA T C AGA A TGAGGGT T A TACA CAGC TC A A AA GGACC T GGCA T T CGACCC T AGT C T T ACT CCCAA T ATGT GT CGAGT T 151 200 Dna. A box A C T GA A C A ACGGT TCT T TGGA T A A CTA CCGGT T GA T CCA AGCT T CCT T GAC T T GCC A ACAAGA A ACCT A T T GAT GGCCA ACT AGGT T CGA AGGA 201 250 Dna. A box Brooker, fig 13. 5 GA C AGAGT TA T CCA CAGTAGA T CGC – 3′ CT GT C A AT AGGT GTCAT C T AGC G 251 275 Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

3′ 5′ 5′ 3′ AT-rich region Dna. A boxes Dna. A proteins bind to Dna. A boxes and to each other. Additional proteins that cause the DNA to bend also bind (not shown). This causes the region to wrap around the Dna. A proteins and separates the AT-rich region. How the origin sequence initiates replication Dna. A protein ATrich region 5′ 3′ 3′ 5′ DNA helicase (Dna. B protein) binds to the origin. Dna. C protein (not shown) assists this process. DNA helicase 5′ 3′ 3′ 5′ DNA helicase separates the DNA in both directions, creating 2 replication forks. 3′ 5′ 3′ Brooker fig 13. 6 Fork 5′ Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

Replication initiation cont. Travels along the DNA in the 5’ to 3’ direction DNA helicase 5′ 3′ 3′ 5′ DNA helicase separates the DNA in both directions, creating 2 replication forks. 3′ 5′ 3′ Fork 5′ Bidirectional replication Brooker fig 13. 6 Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

Schematic side view of DNA polymerase III (bacterial) DNA polymerase catalytic site Thumb 3′ exonuclease site 3′ 5′ Fingers 3′ Palm 5′ Template strand Incoming DNA nucleotides (triphosphates) (d. NTPs) Brooker, fig 13. 8 Copyright ©The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display

Model for how the leading strand lagging strand coordinate at the replication fork Replisome Leading strand 3′ Replication DNA helicase fork Topoisomerase 3′ 5′ Single-strand binding proteins 5′ DNA polymerase III Primosome Region where next Okazaki fragment will be made Primase RNA primer 5′ 5′ 5′ 3′ New Okazaki fragment Older Okazaki fragment Brooker Fig 13. 12 Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

Orientation of lagging strand in the replication bubble

Eukaryotes have hundreds of origins of replication on their (linear) chromosome Chromosome Sister chromatids Origin Centromere (DNA under the kinetochore) Origin Before S phase Brooker, ch 13 During S phase Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display. End of S phase

Replicating DNA of Eukaryotic Chromosomes (Drosophila melanogaster) Fig from i. Genetics, Russell

Bacteria only have one origin on their (circular) chromosome Origin of replication Site where replication ends (a) Bacterial chromosome replication Replication fork Brooker, fig 13. 4 a From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963). Copyright holder is Cold Spring Habour Laboratory Press. 0. 25 μm (b) Autoradiograph of an E. coli chromosome in the act of replication Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display. Replication forks

Replication rate • Eukaryotic DNA replication – – – Typical human chromosome length: 100 million bp Time to replicate a chromosome: minutes to hours Hundreds of origins per chromosome Replicon = ~20, 000 to 300, 000 bp long 500 -5000 bp / minute at each replication fork (slower than bacterial replication; that much harder to “unwind” the DNA for replication). • Bacterial (prokaryotic) replication: – Single circular chromosome (~4. 6 million base pairs [bp]) – Single origin of replication single replicon (“Replication Bubble”)

Requirements of DNA Replication in a complex organism • Very low error rate: – One human cell: 6 billion bp of DNA. A copying error rate of 1 error/million nt 6000 errors with every cell division • Very fast copy rate – E. coli – 1000 nt per minute 3 days to replicate (real life: 20 minutes per cell cycle; 1000 nt per second)

Linear chromosomes (eukaryotic) cannot easily replicate the ends of chromosomes No place for a primer 3′ 5′ Brooker, fig 13. 21 DNA polymerase cannot link these two nucleotides together without a primer.

Linear chromosomes (eukaryotic) must fill in gap left by RNA primer Chromosome gets shorter at the telomeres with each replication if overhang is left. Telomeric repeat sequences 3′ T T A GGG T T A GGG T T A GGG A A TCCCAA TCCCA A TCCCAA T 5′ In humans and most complex organisms, telomerase is only used in continuously dividing stem cells (e. g. spermatogonia stem cells) most cells get shorter telomeres over time (age). What happened to Dolly, the cloned sheep? (she was generated from a skin cell with shorter telomeres, and she aged early) Copyright ©The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display Overhang Brooker, fig 13. 20

How telomerase “finishes” the replication of linear chromosomes Telomere 5′ 3′ 3′ 5′ Eukaryotic chromosome Repeat unit The bindingpolymerizationtranslocation cycle occurs many times T T A G G GT T A GG G T T A G GG T TA G G G 3 CCCA AU C CC A A T C C CAA T Telomerase synthesizes a 6 -nucleotide repeat. Step 1 Binding RNA 3′ 5′ Telomerase G G T T A G G GT T A GG G T T A G G GT T A G A A T C C CAA T This greatly lengthens one of the strands Step 2 Polymerization CC C A A U C C C Telomerase moves 6 nucleotides to the right and begins to make another repeat. T T Step 3 Translocation T T A G G GT T A GG G T T A G G GT T A G G G A A T C C CAA T C C C A A U CCC The complementary strand is made by primase, DNA polymerase, and ligase. T T A G G G T T A G G G 3′ A A T C C CAA T C CC A A T C C C A AU C CC A A U Brooker, figure 13. 22 RNA primer Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display. The end is now lengthened

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Concept Checks • In the Meselson and Stahl experiment, how was switching the bacterial media from N 15 to N 14 important for supporting the Semi-conservative model?