Why Does DNA Use T Instead of U?

Transcription

1 Why Does D Use Instead of U? deoxycytidine C ~100 per day per cell deoxyuridine U roblem: deaminated C is identical to U (and pairs with ) deamination D repair machinery removes all Us from D C U D repair C D uses instead of U to avoid mutations arising from damaged (deaminated) C, which is identical to U Lectures 3-5: ucleic acids & the chemical requirements for replicating information 1. he primary biological roles of nucleic acids 2. he molecular components of D and R a. he primary structure of deoxyribonucleic acid b. he phosphate group in D; equilibrium, acidity, and protonation states c. he sugar group in D; strand orientation and macromolecular chirality d. he bases of D e. he primary structure of ribonucleic acid f. Why does D use deoxyribose? Why? 3. he factors behind D base pairing a. D hybridization as an equilibrium b. he role of hydrogen bonding c. he role of the hydrophobic effect and base stacking 4. he molecular basis of D replication a. D replication; chemical reactions, substrates, and products b. he role of D polymerase: faster and more accurate D replication c. he polymerase chain reaction (CR) and its impact on the life sciences 1

2 D Replication Relies on Base airing D replication D ybridization Equilibrium + B C K eq = [C] [][B] >> 1 (e.g., 10,000 M -1 ) Under typical conditions, K eq >> 1 and there are many more double-stranded molecules than single-stranded molecules What causes D hybridization to be favorable? 2

3 ydrogen Bonding: Matched Base airs + + C 3 Single-stranded: 4 hydrogen bonds ydrogen bonding alone may not strongly favor either side Double-stranded: 4 hydrogen bonds he same # of -bonds are possible on both sides, although scientists continue to debate how these -bonds differ in strength Within a double helix C 3 ydrogen Bonding: Mismatched Base airs ydrogen bonding alone favors the unpaired side for mismatched bases ydrogen bonding alone may not induce D hybridization, but the loss of hydrogen bonds disfavors mismatched pairing + + Single-stranded: 4 hydrogen bonds K eq = ~ 0.01 Within a double helix; no room for water molecules here Double-stranded: 2 hydrogen bonds 3

4 he ydrophobic Effect Water forms an ordered lattice around hydrophobic (oily) surfaces 3 C C 2 2 C C 2 2 C C 2 2 C C 3 ordered water 3 C C 3 C 2 2 C C 2 2 C C 2 2 C 3 C C 2 2 C C 2 2 C C 2 2 C C 3 3 C C 2 2 C C 2 2 C C 2 2 C C 3 rdered waters are disfavored because they are capable of less motion (they have less entropy) Fewer water molecules are ordered when hydrophobic groups are gathered together + disordered water he ucleic cid Bases have ydrophobic Surfaces Rotate 90 o op and bottom surfaces are hydrophobic Water exposure of these hydrophobic surfaces is minimized by stacking the bases In double-stranded D, the bases are largely stacked Bases stacked as in double-stranded D ot accessible to water 4

5 D ybridization is Largely Driven by the ydrophobic Effect o room for water molecules! D hybridization minimizes water-exposed hydrophobic surfaces Lectures 3-5: ucleic acids & the chemical requirements for replicating information 1. he primary biological roles of nucleic acids 2. he molecular components of D and R a. he primary structure of deoxyribonucleic acid b. he phosphate group in D; equilibrium, acidity, and protonation states c. he sugar group in D; strand orientation and macromolecular chirality d. he bases of D e. he primary structure of ribonucleic acid f. Why does D use deoxyribose? Why? 3. he factors behind D base pairing a. D hybridization as an equilibrium b. he role of hydrogen bonding c. he role of the hydrophobic effect and base stacking 4. he molecular basis of D replication a. D replication; chemical reactions, substrates, and products b. he role of D polymerase: faster and more accurate D replication c. he polymerase chain reaction (CR) and its impact on the life sciences 5

6 ow Does D Replicate? Deoxynucleotide triphosphates d d dc 3C d emplate rimer olymerization Extended Base airing Determines Selectivity During D olymerization d C 3 -? d 3C template Base d : dc pyrophosphate he nucleotide capable of forming a base pair with the template is added to the Extended with new group 3C 6

7 D olymerization, South ark-style C - - Comedy Central (don t sue me, please) C - D olymerization in ction I template 3 C d d dc d 7

8 C - D olymerization in ction II template 3 C dc d (dc) d d C - D olymerization in ction III template dc 3 C d d d new group of growing strand pyrophosphate 8

9 C - D olymerization in ction IV template 3 C d d dc d D olymerization in ction V C - template 3 C dc d d d (dc) 9

10 C - D olymerization in ction VI template 3 C dc d d d ote that the new strand is growing in the to direction D olymerase ccelerates Replication dc + Very slow (no observable reaction) dc + D polymerase Fast (~50 bases added per second) 10

11 D olymerization in Cells, C-Style C - - D polymerization in cells requires D polymerase plus several other proteins to unwind double-stranded D and to perform several other essential tasks) nimation by Drew Berry, used with permission D Replication is Extremely ccurate Method Error Rate Selectivity based on hydrogen bonding alone: ~1 in 100 With D polymerase: ~1 in 100,000,000 With all cellular machinery: ~1 in 10,000,000,000 11

12 he olymerase Chain Reaction (CR) olymerase rimers Four ds 3C he CR Cycle Repeat Melt template (heat) Extend s ybridize s (cool) 12

13 CR Exponentially mplifies D ne D template molecule after 25 CR rounds gives 2 25 = 33,554,432 molecules! hermus aquaticus (aq) D olymerase aq D polymerase: operates at 74 C (extension temperature), tolerant of 95 C (melting temperature) hermostable polymerases (and the diversity of life on earth) made CR practical 13

14 CR pplications: Molecular Biology gene of interest can be amplified and inserted into a model organism specific mutation in a proteinencoding gene can be introduced genomic D CR using s that match the ends of the gene CR with mutationcontaining Introduce gene into bacteria Reassemble mutant gene Bacteria use gene to make protein Isolate protein, study its function mutant protein vs. Make mutant protein Make mutant protein; compare properties to wild-type protein wild-type protein CR pplications: IV Detection Because CR can amplify extremely small quantities of D, it can be used as a sensitive method of pathogen detection Blood sample Isolate IV genome (R) CR amplification indicates presence of IV Convert to D CR o CR product indicates no detectable IV levels Sensitivity limit = ~ copies of IV genome 14

15 Key oints: ow D Meets the Requirements for the Blueprint of Life Resist degradation: negatively charged phosphates; no 2 (equilibrium, acidity, K a, pk a, p, enderson-asselbalch) Be recognized by cellular machinery: phosphate groups and bases (ionic bonds and hydrogen bonds) Contain multiple possible structures (bits) at each position: four possible bases per nucleotide ossess redundancy for error correction and replication: base pairing (hydrogen bonding, hydrophobic effect) Knowledge of D replication lead to CR, a key invention Understanding the chemistry of D is crucial to the life sciences 15