Biochemistry of Medicinals I – Nucleic Acids

Instructor: Natalia Tretyakova, Ph.D. 760E CCRB (Cancer Center)Tel. 6-3432e-mail trety001@umn.edu

Lecture: MWF 2:30-3:20 7-135 WDH

Web page: see “Web enhanced courses”

Chapter 1. DNA Structure.

Required reading: Stryer 5th Edition p. 117-125, 144-146, 152, 745-750, 754-762, 875-877) (or Stryer’s Biochemistry 4th edition p. 75-77,80-88, 119-122, 126-128, 787-799, 975-980)

DNA Structure: Chapter outline

1. Biological roles of DNA. Flow of genetic information.

2. Primary and secondary structure of DNA.

3. Types of DNA double helix. Sequence-specific DNA recognition by proteins.

4. Biophysical properties of DNA.

5. DNA topology. Topoisomerases.

6. Restriction Endonucleases. Molecular Cloning

Nucleic Acids

DNA RNA

Central Dogma of Biology

DNA RNA Proteins Cellular Action

transcriptiontranslationDNA

rep

licati

on

(deoxyribonucleic acids) (ribonucleic acids)

Why ?

Questions?

•How is genetic information transmitted to progeny cells?•How is DNA synthesis initiated?•What causes DNA defects and what are their biological an physiological consequences?•What causes the differences between cells containing the same genetic information?

Relevance:

•Cancer: ex. Xeroderma pigmentosum•Genetic diseases: ex., cystic fibrosis, sickle cell anemia,

inborn errors of metabolism•Genetic typing: ex., drug metabolism•Rational drug design: ex., antitumor and antimicrobial

drugs•Biotechnology: ex., growth hormones

The Building Blocks of DNA

-OO

H(OH)

HH

HHO

OP

O

O-

Purine orPyrimidineBase

Phosphate

Pentose sugar

Nucleoside

Nucleotide

1'

2'3'

4'

5'-N-glycosidic bond

DNA and RNA nucleobases

NN

NNH

NH2

NNH

NNH

O

NH2

N

NH

NH2

O

H3C

NH

NH

O

O

Guanine (G)Adenine (A)

Thymine (T)Cytosine (C)

NH

NH

O

O

Uracil (U)

NN

NNH

Purine

N

NH

Pyrimidine

1

2

3

4

5

6

3

2

16

4

57

8

9

(DNA only) (RNA only)

Purine Nucleotides

Pyrimidine Nucleotides

nucleobase (Deoxy)nucleoside

5’-mononucleotide

Adenine (A)

Guanine (G)

Thymine (T)

Cytosine (C)

Uracil (U)

2’-Deoxyadenosine (dA)2’- Deoxyguanosine (dG)2’- Deoxythymidine (dT)2’- Deoxycytidine (dC)Uridine (U)

Deoxyadenosine 5’-monophosphate (5’-dAMP)Deoxyguanosine 5’-monophosphate (5’-dGMP)Deoxythymidine 5’-monophosphate (5’-dTMP)Deoxycytidine 5’-monophosphate (5’-dCMP)Uridine 5’-monophosphate (5’-UMP)

Nomenclature of nucleobases, nucleosides,

and mononucleotides

Structural differences between DNA and RNA

H3CNH

NH

O

O

Thymine (T)

NH

NH

O

O

Uracil (U)

DNA RNA

O

H

HHH

CH2

HO

HOBase

2'-deoxyribose

O

OH

HH

CH2

HO

HOBase

ribose

H

Preferred conformations of nucleobases and sugars in DNA and RNA

HO

O

OH

N

N

NH2

O

Anti conformation

HO

O

OH

N

N

NH2

O

Syn conformation

HO

OH (OH)

HO

BASEHO

O

H (OH)

HOBASE

2' endo (B-DNA)

1'

3' endo (RNA)

3'

1'3'

2' 5'5'

7.0 A

5.9 A

Sugar puckers:

Nucleosides Must Be Converted to5’-Triphosphates to be Part of DNA and

RNA

HOO

OH

OO

OH

PHO

HO

O

OO

OH

P

O

P

OHO

HOO

OH

Base Base

BaseO

O

OH

P

O

P

O

O

OHBase

OH

OP

OHO

HO

Kinase

Kinase

Kinase

Monophosphate

DiphosphateTriphosphate

ATP

ATP

ATP

DNA isArranged5’ to 3’

Connected by

Phosphates

Linking in DNA biopolymer: DNA primary structure

DNA secondary structure – double helix

James Watson and Francis Crick, 1953- proposed a model for DNA structure

•DNA is the molecule of heredity (O.Avery, 1944)

•X-ray diffraction (R.Franklin and M. Wilkins)

•E. Chargaff (1940s) G = C and A = T in DNA

Francis Crick Jim Watson

Watson-Crick model of DNA was based on X-ray diffraction picture of DNA fibres

(Rosalind Franklin and Maurice Wilkins)

Rosalind Franklin

Watson-Crick model of DNA was consistent with Chargaff’s base composition rules

Erwin Chargaff (Columbia University)

G = C and A = T in DNA

DNA is Composed of Complementary Strands

NH

N

N O

NH2

NN

N

H2N

O

HN

N

O

O

NN

N

N NH2

G•C

A•T

DNA

NH

N

N O

NH2

NN

N

H2N

O

HN

N

O

O

NN

N

N NH2

G•C

A•U

RNA

A ::

G :::

T ::

T

C

A

3'

3' 5'

5'

Anti-parallel Strands of DNA

Base Pairing is Determined by Hydrogen Bonding

same size

Base stacking: an axial view of B-DNA

Forces stabilizing DNA double helix

1. Hydrogen bonding (2-3 kcal/mol per base pair)

2. Stacking (hydrophobic) interactions (4-15 kcal/mol per base pair)

3. Electrostatic forces.

right handed helix

• planes of bases are nearlyperpendicular to the helix axis.

•Sugars are in the 2’ endo conformation.

•Bases are the anti conformation.

•Bases have a helical twist of 36º (10.4 bases per helix turn)

• Helical pitch = 34 A

B-DNA

• 3.4 A rise between base pairs

Wide and deep

Narrow and deep

HO

O

OH

N

N

NH2

O

HO

OH (OH)

HO

BASE

1'3'

2'5'

7.0 A

• helical axis passes through

base pairs

23.7 A

DNA can deviate from the ideal Watson-Crick structure

• Helical twist ranges from 28 to 42°

• Propeller twisting 10 to 20°

•Base pair roll

Major groove and Minor groove of DNA

Major groove

Minor groove

NH

N

N O

NH 2

N NN

H 2N

OC-1’C-1’

HN

N

O

O

NN

N

N NH 2

C-1’

C-1’

Major groove

Minor groove

Base BaseTo deoxyribose-C1’ C1’ -To deoxyribose

Hypothetical situation: the two grooves would have similar size if dR residues were attached at 180° to each other

Major and minor groove of the double helix

Wide and deep

Narrow and deep

Major groove

NH

N

N O

NH2

N NN

H2N

O

Minor grooveTo deo

xyrib

ose C-1’

C-1’

HN

N

O

O

NN

N

N NH2

C-1’

C-1’

B-type duplex is not possible for RNA

steric “clash”

O

OH

HH

CH2

HO

HOBase

ribose

H

A-form helix: dehydrated DNA; RNA-DNA hybrids

Top View

Right handed helix

• planes of bases are tilted

20 ° relative the helix axis.

• 2.3 A rise between base pairs

•Sugars are in the 3’ endo conformation.

•Bases are the anti conformation.

•11 bases per helix turn

• Helical pitch = 25.3 A

25.5 A

The sugar puckering in A-DNA is 3’-endo

O

OH (OH)

O

BASEO

O

H (OH)

OBASE

2' endo (3' exo) B-DNA

1'

3' endo (A-DNA)

3'

1'3'

2'

5'

5'

2'

7.0 A

5.9 A

A-DNA has a shallow minor groove and a deep

major groove

N

NH

N

N

O

NH2

NN

H2N

O

To deo

xyrib

ose To deoxyribose

Major groove

Minor groove

B-DNA

N

NH

N

N

O

NH2

NN

H2N

O

To deo

xyrib

ose To deoxyribose

Major groove

Minor groove

Helix axis

A-DNA

• •

Z-form double helix: polynucleotides of alternating purines and pyrimidines (GCGCGCGC) at

high salt

Left handed helix

• Backbone zig-zags because sugar puckers alternate between 2’ endo pyrimidines and 3’ endo (purines)

• Bases alternate between anti (pyrimidines) and syn conformation (purines).

•12 bases per helix turn

• Helical pitch = 45.6 A

• planes of the bases are tilted 9° relative the helix axis.

• Flat major groove• Narrow and deep minor groove

18.4 A

• 3.8 A rise between base pairs

Sugar and base conformations in Z-DNA alternate:

N

N

NH2

ON

HN

NN

O

H2NHO

OH

HO

HO

O

H

HO1' 3'

1'3'

2'

5'

5'

GC

5’-GCGCGCGCGCGCG3’-CGCGCGCGCGCGC

C: sugar is 2’-endo, base is antiG: sugar is 3’-endo, base is syn

Biological relevance of the minor types of DNA secondary structure

•Although the majority of chromosomal DNA is in B-form, some regions assume A- or Z-like structure

• Runs of multiple Gs are A-like

•The upstream sequences of some genes contain 5-methylcytosine = Z-like duplex

N

NH

NH2

O

5-methylcytosine (5-Me-C)

H3C

• RNA-DNA hybrids and ds RNA have an A-type structure

• Structural variations play a role in DNA-protein interactions