Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins - PowerPoint PPT Presentation

Loading...

PPT – Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins PowerPoint presentation | free to download - id: 459b78-ZGVhZ



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins

Description:

Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins The edges of base pairs displayed to DNA major and minor groove – PowerPoint PPT presentation

Number of Views:169
Avg rating:3.0/5.0
Slides: 39
Provided by: tret4
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins


1
Hydrogen bond donors and acceptors in DNA grooves
facilitate its recognition by proteins
The edges of base pairs displayed to DNA major
and minor groove contain potential H-bond donors
and acceptors
O
N
h
n
h
o
n Nitrogen hydrogen bond acceptor o Oxygen
hydrogen bond acceptor h Amino hydrogen bond
donor
2
Hydrogen bond donors and acceptors on each edge
of a base pair
3
Structural characteristics of DNA facilitating
DNA-Protein Recogtnition
  • Major and major groove of DNA contain sequence-
  • dependent patterns of H-bond donors and
    acceptors.
  • Sequence-dependent duplex structure (A, B, Z,
    bent
  • DNA).
  • Hydrophobic interactions via intercalation.
  • Ionic interactions with phosphates.

4
Groove binding proteins and drugs
5
Triple helix and Antigene approach
Hoogsteen base pairing parallel Reversed
Hoogsteen antiparallel
6
Biophysical properties of DNA
  • Facile denaturation (melting) and re-association
    of the duplex
  • are important for DNAs biological functions.
  • In the laboratory, melting can be induced by
    heating.

Single strands
T
duplex
  • Hybridization techniques are based on the
    affinity of complementary
  • DNA strands for each other.
  • Duplex stability is affected by DNA length,
    GC base pairs, ionic strength, the presence of
    organic solvents, pH
  • Negative charge can be separated by gel
    electrophoresis

7
Separation of DNA fragments by gel
electrophoresis
Polyacrylamide gel
  • DNA strands are negatively charged
  • migrate towards the () electrode (anode)
  • Migration time ln (number of base
  • pairs)

8
DNA Topology DNA has to be coiled to fit inside
the cell
Organism Number of base pairs Contour length, ?m
E. Coli bacteria 4,600,000 1,360
SV40 virus 5,100 1.7
Human chromosomes 48,000,000- 240,000,000 1.6 8.2 cm
DNA polymers must be folded to fit into the cell
or nucleus (tertiary structure).
9
DNA Topology
10
DNA Topology linking number
11
Consider a 260 bp B-duplex
12
Connect the ends to make a circular DNA
Tw 260/10.4 25
13
(No Transcript)
14
(No Transcript)
15
(No Transcript)
16
An electron micrograph of negatively supercoiled
and relaxed DNA
Stryer Fig. 27.20
17
Organization of chromosomal DNA
Chromosomal DNA is organized in loops (no free
ends) It is negatively supercoiled 1 (-)
supercoil per 200 nucleotides
145 bp duplex
Histone octamer (H2A, H2B, H3, H4)2
H1 is bound to the linker region
18
Enzymes that control DNA supercoiling DNA
Topoisomerases
Change the linking number (Lk) of DNA duplex by
concerted breakage and re-joining DNA strands
Topoisomerase enzymes
Topoisomerases I Relax DNA supercoiling by
increments of 1 (cleave one strand)
Topoisomerases II Change DNA supercoiling by the
increments of 2 (cleave both strands) Usually
introduce negative supercoiling
19
Human DNA Topoisomerase I DNA side view
20Å
Stryer Fig. 27.21
20
Mechanism of DNA Topoisomerases I
723
OH
P-Topo
? Wr 1
21
Drugs that inhibit DNA Topoisomerase I
  • Camptothecin, topotecan and analogs
  • Antitumor activity correlates with interference
    with topoisomerase activity
  • Stabilizes topoisomerase I-DNA intermediate,
    preventing DNA strand re-ligation
  • Used in treatment of colorectal, ovarian, and
    small cell lung tumors

22
Enzymes that control DNA supercoiling DNA
Topoisomerases
Change the linking number (Lk) of DNA duplex by
concerted breakage and re-joining DNA strands
Topoisomerase enzymes
Topoisomerases I Relax DNA supercoiling by
increments of 1 (cleave one strand)
Topoisomerases II Change DNA supercoiling by the
increments of 2 (cleave both strands) Usually
introduce negative supercoiling
23
Topoisomerases II
Most of Topoisomerases II introduce negative
supercoils (e.g. E. coli DNA Gyrase) Require
energy (ATP) Each round introduces two
supercoils (? Wr - 2) Necessary for DNA
synthesis Form a covalent DNA-protein complex
similar to Topoisomerases I
24
Yeast DNA Topoisomerase II
Stryer Fig. 27.23
25
Topoisomerase II - mechanism
Stryer Fig. 27.24
26
Drugs that inhibit bacterial Topoisomerase II
(DNA gyrase)
Interfere with breakage and rejoining DNA ends
Inhibit ATP binding
27
Enzymes that cut DNA exonucleases
5
OH
A
5
3
5
3
dNMPs
Degrade DNA in a stepwise manner by removing
deoxynucleotides in 5 ? 3 (A) or 3 ? 5
direction (B) Require a free OH Most
exonucleases are active on both single- and
double-stranded DNA Used for degrading foreign
DNA and in proofreading during DNA synthesis
H
HO
B
3
28
DNA Endonucleases
Cleave internal phosphodiester bonds resulting
in 3-OH and 5-phosphate ends
3-OH
5
5-P
3-OH
5-P
some endonucleases cleave randomly (DNase I, II)
Type II Restriction endonucleases are highly
sequence specific
EcoRI recognition site
Palindromic site (inverted repeat)
RE are found in bacteria where they are used
for protection against foreign DNA
29
Recognition sequences of some common restriction
endonucleases
30
DNARestrictionEnzyme EcoR V
31
Applications of Restriction Endonucleases in
Molecular Biology
  • DNA fingerprinting (restriction fragment length
    polymorphism).
  • 2. Molecular cloning (isolation and
    amplification of genes).

32
Southern blotting
33
Restriction fragment length polymorphisms are
used to compare DNA from different sources
34
DNA Ligase
AMP PPi
O
O
P
O
OH
O-
Forms phosphodiester bonds between 3 OH and 5
phosphate Requires double-stranded DNA
Activates 5phosphate to nucleophilic attack by
transesterification with activated AMP
35
DNA Cloning recombinant DNA technology
36
Human Genetic Polymorphisms
  • Human genome size 3.2 x 109 base pairs
  • 30,000 genes
  • 2-4 of total sequence codes for proteins
  • Human genetic variation
  • ? 1 sigle nucleotide polymorphism (SNP) per 1,300
    bp

37
Examples of genetic polymorphisms of drug
metabolizing enzymes
 Enzyme substrate examples DNA regions
involved cytochrome 2B6 cyclophosphamide exons
1,4,5, and 9 tamoxifen benzodiazepines cyto
chrome 2D6 debrisoquine internal base changes
cytochrome 1A2 caffein 5' flanking
region phenacetin N-acetyltransferase aromati
c amines
38
DNA Structure Take Home Message
  • Genetic information is stored in DNA.
  • DNA is a double stranded biopolymer containing
    repeating units of nitrogen base, deoxyribose
    sugar, and phosphate.
  • DNA can be arranged in 3 types of duplexes which
    contain major and minor grooves.
  • DNA can adopt several topological forms.
  • There are enzymes that will cut DNA, ligate DNA,
    and change the topology of DNA.
  • Human genome contains about 3.2 billion base
    pairs. Inter-individual differences are observed
    at about 1 per 1,000 nucleotides.
About PowerShow.com