DNA Metabolism

DNA metabolism here means (all?) of thechemical processes that DNA undergoes.

Comment on “stabile storage” and newcombinations. (Recombination)

E. coli: archetypal organism for early studiesof DNA metabolism; its DNA related genesare shown on the next page:

Fig. 25-11

I. DNA Replication

Conventions: Name bacterial genes with three lowercase

italicized letters: dnaWhen multiple genes influence same process, add

italicized upper case: dnaBBacterial gene products; no italics, 1st letter

capitalized: DnaB

Template concept: 1. How can you make a copy of a molecule?2. Complementary Hydrogen bonding

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A. DNA replication follows a set of rules1. Semiconservative (doesn’t mean a reddish-purple state)

Evidence: Messelson-Stahl

Fig. 25-2

Grow cells 1st on 15N. [See (a)]Get cells ready for synchronous division, then...Switch to all 14N growth medium.Isolate some of the DNA. [See (b)]Grow though one more cell cycle on 14N medium. [See (c)]

The interpretation pretty simple: i.e., conservative hypothesis is disproved.

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2. Begins at an origin (Ori sites), usually bidirectionallyevidence: 3H labeled DNA.

(Why not 14C?)Fig. 25-3

Would unidirectional copying look different in thisexperiment?

3. Always goes 5'ÿ3', and

4. is therefore semi-discontinuous (Okazaki Fragments)a) leading strandb) lagging strand

Fig. 25-44

B. DNA is degraded by nucleases1. Endo (Remember restriction enzymes from 581?)2. Exo: Specific enzymes normally degrade with

specific polarity (either 5'ÿ3' or 3'ÿ5').

C. DNA is synthesized by DNA polymerasesLonger name: “DNA dependent DNA polymerases.”0. Template requirement!1. Early work on DNA polymerase I (pol I, coded by polA)2. Primer requirement3. 2 active site locations: insertion & post-insertion (25-5)4. Processivity

See Fig. 25-5 on next pages5

Example of Mg2+ at pdb: 1d5a

Note alkoxide ion.

See animation 2501?

Fig. 25 c) is from pdb: 4ktq

Go to: http://www.dnai.org/a/index.html then “Copying the Code”then “Putting it together.”

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D. Replication is very accurate 1. Overall, ~ 1 error per 109 to 1010 bases added, which

is 1 error'103 to 104 replications (Why, tautomers,see Fig. 8-9, next page),

but DNA polymerase initial error rate proper is ~1error'104 to 105.

This is pretty good. Active site size/shape excludesmany incorrect base paring options (Fig. 25-6).

Why the discrepency? a) 3'ÿ5' exonucleaseb) additional enzyme(s) that deal with mismatch issues

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Tautomers of U

2. Base-paring geometry at the active site. Fig 25-6

3. 3' ÿ 5' exonuclease activity of the polymerase:proofreading , see next page, Fig. 25-7 (But reverse transcriptase?) a) Not just reverse rxn. No PPi involved.b) This yields a 100-1000 enhancement in fidelity.

This gets us to an error rate of 1 in 106 to 108. Westill have other factors to identify to understand the overalllow rate of one in 109to 1010. “Other factors” includemismatch repair system described later in the chapter.

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3' ÿ 5' exonuclease activity

Fig. 25-7

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pdb file 1nkb:

“A Bacillus DNApolymerase I productcomplex bound to aguanine-thyminemismatch after threerounds of primerextension, followingincorporation of dCTP,dGTP, and dTTP.”

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E. E. coli has at least Five DNA polymerases!Klenow

fragment ofE. colipolymerase I

5' ÿ 3'exonucleaseactivity hasbeen.

Why wouldPol I havethis activity?

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1. Could DNA polymerase I be the primary enzymethat 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’sdo the calculation!

b) Does the processivity (define) seem consistent? c) Are pol I minus mutants viable? (Knockout bacteria?)

2. Pol I likely involved “clean-up” activities associatedwith DNA metabolism. Nick translation. Fig. 25-8

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3. Pol II acts in recombination.4. Pol III is the main enzyme responsible for

recombination. pdb file: 2pol Note: this shows onlythe 2 β subunits.

What fits in the doughnut?

Ponder evolution of theβ subunit briefly.

See Table 25-2

5. Pol IV & V are activein DNA repair.

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F. DNA replication requires many enzymes andprotein factors

Replicase System (replisome) List:1. helicases2. topoisomerases3. DNA binding proteins (stabilize separated strands)4. primases (RNA) 5. DNA ligases (deal with sealing final connections

after primers are removed & resulting gaps filled in.Fig. 25-9 a)Labeling error???

Animation, again?:http://www.dnai.org/a/index.html Then Copying the Code, then putting it togetherclamp-loading action: Then CD animation 2502?

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DNA Metabolism (part II)

G. E. coli chromosome replication proceeds instages Comment on initiation/elongation/termination.

1. Initiation (site on DNA [oriC] & $ 10 proteins involved)

a) oriC: 245 bp, contains highly conserved sequencesb) DNA unwinding elements (DUE) are usually A'T rich.c) Eight 9 bp repeats w/ high affinity for DnaA (initiator

protein) See Fig 25-10. R & I sites bound by DnaA. Consensus sequence?

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Cast of protein characters: (Table 25-3)

c) DnaA binding induces right-handed supercoiling (whichtype?) that pops open the A'T rich DUE region.

AAA+ ATPase family = ATPases associated w/ diverse cellular activities

d) DnaB (helicase lavendar) then binds (DnaC green/ATP-dependent) to help open DNA. See Fig 25-11, below:

e) Methylation of N6 of A in GATC sequences prior toinitiation. oriC ~ 12 fold enriched in GATC. Is thenascent strand methylated? Does this provide a meansfor regulating initiation?

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2. Elongationa) Leading strand (Prime and forget?)b) Lagging strand (repeated priming is needed) Fig. 25-12,

next page.

Remember logic? Find 3'-OH. Review Fig. 25-5?Nick translation and ligase activities?

Fig. 25-12c) Clamp loader Fig. 25-13.

d) How are primers at start of Okazaki fragments dealt with?

Fig. 25-16 DNA ligase mechanism

Summary comments about the replisome17

3. Terminationa) Multiple copies of 20 bp sequence, Ter. Fig. 25-17b) Tus protein binds to Ter sites on DNA, forming a tight

complex. (Tus = terminus utilization substance) c) Each Tus-Ter complex only stops elongation from one

direction. (If the two sides of the theta don’t arrive at thesame time.) Fig. 25-18

Note directions of action.

d) The 2nd arriving fork stops at the first (arrested) fork. (Because of size of replisome, there is a space. Remaining bases filled in ?) Eventual resolution of withslower copying complex encounter with Ter-Tus notcompletely understood, but see Fig. 25-18 regardingtopological resolution.

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H. Replication in Eukaryotic Cells is Similar butMore Complex

1. General patterns same as prokaryotes, but eukaryoteshave a more formally defined cell cycle, andreplication must be synched up with this.

2. Origins of replication are less clearly defined ineukaryotes relative to bacteria.

3. Eukaryotes usually have many ori equivalent sites. (If we only had one per “average” chromosome, itwould take 500 hours to replicate. Cell divisiontime?) See pre-replicative complex (Fig. 25-19)

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4. Re. Kinetics, the rate of replication fork movement ineukaryotes (50-ish nucleotides/sec) is about 20 timesslower than in prokaryotes.

5. Like bacteria, eukaryotes have multiple DNApolymerases.a) DNA polymerase α (most responsible for replication)b) DNA polymerase δc) DNA polymerase ε

6. Because eukaryotic nuclear DNA is arranged inlinear chromosome, eukaryotes have telomeres at theend of each chromosome. More on these nextchapter.

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I. Viral DNA Polymerases & AntiviralTherapy

1.Logic: a) find something in the pathogen that is biochemically

different from the host. b) find chemical that interacts w/ (inhibits) pathogen protein

more strongly than host protein.

2. Example: acyclovir (Selective at 2 steps)a) phosphorylated in cells infected with herpes virus.

b) Why in those cells? Virus codes for a thymidine (?)kinase that has 200 times higher affinity (KM) foracyclovir than does the equivalent host enzyme.

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c) The phosphorylated product (acyclo-GMP) is convertedto acyclo-GTP by cellular kinases (no selection here?)

d) Finally, acyclo-GTP interacts more strongly with viralDNA polymerase than host DNA polymerase:i) as an inhibitor (KI) & ii) as a substrate to form dead end products (KM)

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DNA Metabolism (part III)

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II. DNA RepairGeneral approach: Something about the damage is

structurally (chemically) different than undamaged DNA. Living things have evolved systems to detect and repairthese differences.

A. Mutations are linked to cancer.

1. Preliminary comment on types of mutations.Google: “McGill University PKU mutation map” or http://www.pahdb.mcgill.ca/?Topic=Information&Section=MutationMap&Page=0

At the site, look at the legend to survey the different types of mutationsthat have been found in the PAH gene. Pick a few; analyze in more detail. (I224M!, W187X#, P89fs see legend) Have a copy of the genetic code handy.

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Fig. 27-7mutation types:silentmissensenonsenseother

2. 90% of carcinogens are mutagens. This correlationallows pre-screening of new compounds todetermine if they are likely to cause cancer. Amestest fig. 25-20

a) background mutation rateb)-d) decreasing [mutagen]

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B. All cells have multiple DNA repair systems.See Table 25-5

1. Mismatch repair a) How to decide what is right, what mismatching? (F. 25-21)b) MutS, L, and H (Fig. 25-22) initiate processc) exonuclease (there’s a free end now), DNA polymerase,

and DNA ligase complete the repair (Fig. 25-23)

Fig. 25-21

Methylation let’s us distinguish the old from new strand

Fig. 25-22

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Cleave new (non-methylated) strand

Fig. 25-23 Completing mismatch repair

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2. Base-Excision repaira) Cleave glycosyl bond to remove base, b) then fill in as in fig. 25-24

3. Nucleotide excision repair: Employed at largedistortions in DNA. Fig. 25-25a) Excise baseb) remove 13-mer or 29-merc) fill in with polymerase

4. Direct repaira) Photolyases (see next page)b) O6-methylguanine demethylase (not really an enzyme,

but MW ~ 60,000)

Fig. 25-2628

C. The interaction of Replication Forks with DNADamage Can Lead to Error-Prone TranslesionDNA Synthesis (TLS) (Whew!)

0. The repair processes considered so far require onestrand to be intact & undamaged. Now lets see whatcan be done when both strands are damage, or onestrand is gone.

1. Backgrounda) What things interact to cause evolutionary changes?

Consider long time frame. Evolutionary scale.

&

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b) What may have forced human evolution in east Africa? See: http://www.ldeo.columbia.edu/~peter/site/Papers_files/Climates-HumanEvolutionFinal.pdf

2. What is likely to happen to currently existingorganisms if the environment changes rapidly?

3. The answer to #2 is where error-prone translesionrepair (SOS system) becomes important. Fig. 25-29a) SOS system can read through blocked areas and fill in

regions where a complete template is not present.b) This system is inherently error-prone. “Things have

gotten so bad I’ll try anything to copy my genome!”c) If the environment is changing rapidly, mutations have a

higher probability of providing variant offspring that arebetter suited to the changing environment.

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Fig. 25-29 How DNA damage can impact replication.

4. In any damage site in DNA where base pairing is notpossible, errors are more likely to happen.

5. See Table 25-6 for a list of proteins involved in theSOS system.

6. Mammals have a TLS-like system:

a) but because of where these proteins work (and alsobecause of other associated activities), their error ratesare not as high as those in the bacteria SOS system.

b) Humans have more than 130 genes (haploid?) thatfunction mainly in repair of DNA.

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D. Comments on Box 25-1, DNA repair & cancer

1. Xeroderma pigmentosum (XP)

2. Back to evolution: “small, furry, nocturnal animalswith little need t repair UV damage.” Relates to lackof back-up pyridine dimer repair systems in allplacental mammals.

3. HNPCC: hereditary non-polyposis colon cancer. This can arise through mutations in 5 different genesinvolved in mismatch repair.

a) Founder mutations?b) Italian origin, comment on de novo mutations.

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4. BRCA1 & BRCA2 genesa) Comment on a disease having a good PR program?b) Genes are involved in DNA recombination/repair.c) Different ways to “have” the BRCA genes?d) Just impacting breast cancer odds?e) Just impacting women?f) Comment on dominant vs. recessive inheritance?

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DNA Metabolism part IV

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III. DNA Recombination

Introductory comment: Three main types ofrecombination1. Homologous genetic recombination2. Site specific recombination3. DNA transposition4. Other5. FunctionsIntegration into other DNA metabolic processes.

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A. Bacterial Homologous Recombination is (also)a DNA repair function (Why would it not havethe same significance in generating geneticdiversity that it has in eukaryotes? But:transformation and conjugation [rare in wild])

1. Helps repair DNA damage. Term: recombinational DNA repair.

a) DNA part of general process shown in Fig. 25-30

Fig 25-30

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b) 5'-end processingStep ì in 25-30

Fig. 25-31

RecBCD complex: motors (helicases) and a 3'ÿ5'exonuclease activity

Note: chi site is a specific DNA sequence:(5')GCTGGTGG

c) RecA is an unusual protein (lots of different activities)

Here (Fig. 25-32 a) RecA works to achieve strand

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invasion. Step í from Fig. 25-30.

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Fig. 25-31 b) & c)

d) After strand invasion has occured, RuvABpromotes branch migration (step î in Fig. 25-30. Also see Fig. 25-33a for a more detailed look.)

e) The Holliday intermediate is then cleaved by aspecialized endonuclease, RuvC. (step ï in Fig.25-30. Also see Fig. 25-33b for more detail.)

f) After recombination is completed, the replicationfork is re-started by a system that achieves origin-independent restart.

2. Increases genetic diversity

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Inserting pieces of DNA using biotechnology (1:12):http://www.youtube.com/watch?v=8rXizmLjegI

Generalized recombination: Birds do it, bees do it, even plasmids??? (4:58)http://www.youtube.com/watch?v=XalBdrNvFuo

Animation (1:39) demonstrating how genetic diversity is enhancedby recombination in meiosis:http://www.youtube.com/watch?v=f18U__0nBxQ&feature=related

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B. Eukaryotic Homologous Recombination IsRequired for Proper Chromosome Separationduring Meiosis See Fig. 25-34 & 25-35.

animations:

1. Intro: a. Are eukaryotes haploid or diploid?b. Homologous recombination does have repair functions

(stalled forks).c. However, it occurs with highest frequency in meiosis

Meiosis for the chemists: short- http://www.youtube.com/watch?v=D1_-mQS_FZ0 (1:48) longer- http://www.youtube.com/watch?v=vA8aMpHwYh0 (2:57)

Longer, more context but will subject you to graphic images of prairie dog foreplay: http://www.youtube.com/watch?v=mKWxeMMFTEU (11:47)

Biology students: I’m not a biologist. If you have better suggestions, please let me know.

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2. Key stages in meiosis Fig. 25-34

a) When spindle fibers start to pull on centromeres, physicallinks between chromosomes cause a tension to develop inthe spindle fibers (before they actually start to separatethe chromosomes). During the 1st meitotic division, themain links are homologous recombination complexes. See the chiasmata in Fig. 25-35, step ï product, a, b, & c.

b) If the chromosomes are not linked firmly enough to causesufficient tension, the process stalls. Spindle fibersdissociate from the centromere, and after some period thecell “tries again.” See Box 25-2 re. imperfect chromosome segregation.

c) Note: 4 genetically different gametes produced (Fig. 25-34, bottom line)

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Homologous recombination:http://www.youtube.com/watch?v=SAqGKWz109M (1:55)

Holliday intermediate resolution: (Which video is more effective?)http://www.youtube.com/watch?v=BhJf9MHHmc4 (1:53)http://www.youtube.com/watch?v=gQFKdA3VgEg&feature=related (0:42)

3. Summary of functions of homolgous recombinationa) DNA damage repairb) Link to increase odds of proper chromosome segregationc) Enhance genetic diversity in gamete population.

Independent assortment of chromosomes (comment onlinkage) is also important in generating geneticallydiverse gametes, Fig. 25-37.

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Fig. 25-36

How many possibilities for 23 chromosomes?

C. Recombination during Meiosis is Initiated withDouble-Strand Breaks.

1. General pattern is shown in Fig. 25-35. It has manysimilarities to bacterial homologous recombinationfor DNA repair (see Fig. 25-30).

2. Extending 3' ends initiate exchange, serving asprimers to fill in complementary DNA seqences(shown in purple in Fig. 25-35).

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3. Both of the Holliday intermediate resolution productsshown after step ð are observed in vivo.

D. Site-Specific Recombination Results in PreciseDNA Rearrangements

1. Very different from homologous recombination inthat it is limited to specific DNA sequences.

2. Functions/components somewhat species specific.

3. Components:a) Specific DNA sequenceb) Recombinase

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i) Tyr at active site (see Fig. 25-37 re. lambda enzyme)ii) Ser at active site

Fig. 25-37aFig. 25-37b

4. Site specific recombination sequences are usually notperfectly pallendromic (symmetrical). Outomes:

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E. Transposable Genetic Elements Mover fromOne Location to Another

Bacteria have two types:1. Simple (a.k.a., insertion sequences) contain

a) DNA sequenceb) gene(s) for proteins to catalyze process

2. Complex contain a) & b) and some additional gene(s)

Important: Mechanism for gene duplication. Fig. 25-40

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F. Immunoglobulin genes assemble byrecombination Derived from a transposition system?0. Antibody structure molecule Fig. 5-21

1. The human genome contains ~29,000 genes.2. The human immune system can produce millions (1.5

× 107) of different antibody molecules(immunoglobulins, Ig’s).

3. How is this possible???See Fig. 25-42 (cassettes) and 25-43

Fig. 25-42

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Light chain Math:

300 V’s × 4 J’s = 1,200 possible V-J’s

Recombination fuzzy, causes ~2.5 multiplier:

1,200 × 2.5 = 3,000 light chain combinations

Similar for heavy chain gives 5,000 combinations.

For whole IgG: 3,000 × 5,000 = 15,000,000 options

Evidence for immune system strength importance?49