DNA Metabolism. I. DNA Replication. A. Template concept: 1. How can you make a copy of a molecule? 2. Complementary Hydrogen bonding

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1 DNA Metabolism I. DNA Replication A. Template concept: 1. How can you make a copy of a molecule? 2. Complementary Hydrogen bonding B. DNA replication follows a set of fundamental rules 1. Semiconservative (does not mean a reddish-purple state) Do Problems 1 & 2, p Begins at an origin (Ori sites), usually bidirectionally 3. Always goes 5' 3', and 4. is therefore semi-discontinuous (Okazaki Fragments) a) leading strand b) lagging strand 1

2 C. DNA is degraded by nucleases 1. Endo 2. Exo D. DNA is synthesized by DNA polymerases A longer name would be DNA dependent DNA polymerases. (Template requirement!) 1. DNA polymerase I (pol I, coded for by pola in E.coli) 2. Primer requirement 3. Processivity Note alkoxide ion. See animation 2501? E. Replication is very accurate ~ 1 error per 10 to 10 bases added, which is 1 error per 10 to 10 replications 2. Base-paring geometry Visualize steric/bonding elements at the active site. 3. 3' 5' exonuclease activity of the polymerase: proofreading (Reverse transcriptase?) This yields a enhancement in fidelity. pdb file 1nkb: A Bacillus DNA polymerase I product complex bound to a guanine-thymine mismatch after three rounds of primer extension, following incorporation of dctp, dgtp, and dttp. 2

F. E. coli has at least Five DNA polymerases! Klenow fragment of E. coli polymerase I 1. Could DNA polymerase I be the primary enzyme that catalyzes replication of the E. coli chromosome?

3 F. E. coli has at least Five DNA polymerases! Klenow fragment of E. coli polymerase I 1. Could DNA polymerase I be the primary enzyme that catalyzes replication of the E. coli chromosome? Lets look at some data: a) How long would it take for pol I to make a copy of the E. coli chromosome (asume bidirectional copying)? Let s do the calculation! b) Does the processivity (define) seem consistent? c) Are pol I minus mutants viable? 2. Pol I appears to be involved clean-up activities associated with DNA metabolism. Nick translation. Fig Pol II acts in recombination. 4. Pol III is the main enzyme responsible for recombination. pdb file: 2pol Note: this shows only the 2 á subunits. What fits in the doughnut? See Table 25-2 Animation: Then Copying the Code, then putting it together clamp-loading action: CD animation Pol IV & V are active in DNA repair. 3

4 G. DNA replication requires many enzymes and protein factors. 1. topoisomerase 2. DNA binding proteins 3. primases (RNA) 4. DNA ligases Replicase List: H. Replication of the E. coli chromosome proceeds in stages 1. Initiation (at least 10 proteins involved) a) DNA unwinding elements (DUE) are usually A T rich. b) 9 bp repeats w/ relatively high affinity for DnaA (initiator protein) See Fig c) DnaA binding induces right-handed supercoiling (which type?) that pops open the A T rich DUE region. d) DnaB (helicase) then binds (DnaC-dependent) to help open DNA. See Fig 25-12: 6 e) Methylation of N of A in GATC sequences prior to initiation. OriC ~ 12 fold enriched in GATC. Is the nascent strand methylated? Does this provide a means for regulating initiation? 2. Elongation a) Leading strand (Prime and forget?) b) Lagging strand (repeated priming is needed) See Fig Nick translation and ligase activities? c) Clamp loader details in Fig

5 3. Termination a) Multiple copies of 20 bp sequence, Ter. b) Tus protein binds to Ter sites forming a relatively tight complex. c) Each Tus-Ter complex only stops elongation from one direction. (If the two sides of the theta don t arrive at the same time.) Fig nd d) The 2 arriving fork stops at the first (arrested) fork. (Because of size of replisome, there is a space. Remaining bases filled in?) e) Sorting out the tangles: topoisomerase IV. Fig Video aside on common ancestry: 1. Endogenous retroviral sequences: 2. Human chromosome 2: Comments on Acyclovir (re. p. 992:) 5

6 II. DNA Repair Ames test fig A. Mutations are linked to cancer. 90% of carcinogens are mutagens B. All cells have multiple DNA repair systems. See Table Mismatch repair (How to decide what is right and what is mismatching?) Fig Base-Excision repair a) Cleave glycosyl bond to remove base, b) then fill in as in fig Nucleotide excision repair: Employed at large distortions in DNA. a) Excise base b) remove 13-mer or 29-mer c) fill in with polymerase 4. Direct repair a) Photolyases 6 b) O -methylguanine demethylase (not really an enzyme, but MW ~ 60,000) 6

7 III. DNA Recombination A. Three main types of recombination 1. Homologous genetic recombination 2. Site specific recomb 3. DNA transposition 4. Other 5. Functions Integration into other DNA metabolic processes. B. Homologous genetic recombination has several functions 1. Helps repair DNA damage 2. Physical link that aids in chromosome separation (meiosis) 3. Increases genetic diversity animations demonstrating how genetic diversity is enhanced by recombination in meiosis: 7

8 0nBxQ&feature=related C. Recombination during meiosis is initiated with double-strand breaks See Fig through animations: Interactions: 1. Branch migration 2. Nuclease-DNA polymerase activity Double strand break repair model: Holliday intermediate resolution: D. Recombination requires a host of enzymes and other proteins 1. RecA protein enhances rates of DNA pairing, branch migration, etc. (Note unusual structure: Fig , needed for spooling to drive strand exchange? Fig ) 2. RecBCD enzyme has helicase and nuclease activities 8

9 E. All of DNA metabolism works to fix stalled replication forks F. Site-specific recombination results in precise DNA rearrangements 1. Unique DNA sequence 2. Recombinase that recognized the specific DNA sequence 3. Certain bacteriophage (ex.: lambda) human Ig? s? 4. Structure of DNA complexed to Cre recombinase (Fig 25-40) 5. See lambda integration, Fig G. Complete chromosome replication can require site-specific recombination. Fig Resolution of a dimeric chromosome. H. Transposable genetic elements move from one location to another 1. Simple = insertion sequences 9

10 a) DNA sequences necessary b) Genes for transposases 2. Complex transposons have additional genes related to the process beyond those noted in #1, above. I. Immunoglobulin genes assemble by recombination 1. The human genome codes for no more than ~30,000 genes. 2. The human immune system can produce millions of different antibody molecules (immunoglobulins, Ig s). 3. How is this possible??? See Fig (cassettes) and

DNA metabolism. DNA Replication DNA Repair DNA Recombination

DNA metabolism. DNA Replication DNA Repair DNA Recombination DNA metabolism DNA Replication DNA Repair DNA Recombination Chutima Talabnin Ph.D. School of Biochemistry,Institute of Science, Suranaree University of Technology Central Dogma or Flow of genetic information