DNA Structure, Topology, Recognition

DNA Structure

phosphodiester bonds

hydrogen bonding

double helix

Recognition of DNA by Proteins

DNA binding domains

hydrogen bonding

DNA Topology

linking number

supercoiling

topoisomerases

Higher Order Structure

histones

nucleosomes

chromatin

Information pathways - the central dogma

DNA is an informational molecule

within genes

outside of genes

polymer of _________________

DNA is an informational molecule

• sequence of bases specifies genetic information

  • within genes

    • nucleotides specify the amino acid sequences of proteins

  • outside of genes

    • regulatory sequences direct:

      • DNA replication

      • mRNA synthesis

        • when, where, how much?

DNA is a polymer of deoxyribonucleotides

Deoxyribonucleotides

Nucleotides are linked by _____________ bonds

what direction polarity?

bases remain available for __________ ________

Common hydrogen bonds in biological systems

bond length?

Angled hydrogen bonds are weaker why?

2.6-3Å bond length

Angled hydrogen bonds (due to constraints in protein structures, for example, are weaker.

Hydrogen bonding mediates _____________________- in DNA

Hydrogen bonding mediates base-pairing interactions in DNA

structure of dna

6 points regarding strand interactions, location of backbone vs bases, base pairing, how information is copied

1. Two strands of DNA– held together by base-pairing interactions

2. the DNA strands are antiparallel

3. Sugar-phosphate backbone is on the outside 

4. the bases are stacked on the inside

5. Base pairs are purine-pyrimidine

   1. G pairs with C

   2. A pairs with T

6. Complementarity allows information to be copied

dna double helix

how is it stabilized?

hydrogen bonding + stacking interactions

Structural variation in DNA

A Form DNA:	B Form DNA:	Z Form Helix:
• DNA-RNA, RNA- RNA helix • found in solution • 11 bp per turn • right-handed	• most stable • found in solution • 10.5 bp per turn • right-handed	• left-handed

Denaturation of DNA

tm = temperature at which 50% of the DNA is random coil (not helical)

• Here, tm < ™

  • So, %GC < %GC

    • Why is GC more stable?

As measured by UV absorbance, denaturation → __________?

• due to _____________ = what effect?

High temperature or pH

tm increases linearly with number of GC bonds

RNA Structure

single strand helix, hairpin double helix (secondary structure)

An example of RNA structure: RNase P

Base pairs in RNA:

A-U, G-C

AND

G-U

DNA sequences are recognized by DNA binding proteins

Sequence-specific DNA recognition is key to what?

How are specific DNA sequences recognized?

Sequence-specific DNA recognition is key to carrying out the steps in information transfer

How are specific DNA sequences recognized?

• hydrogen bonding

• major groove interactions

Hydrogen bonding opportunities in double helical DNA

Hydrogen bond acceptors (🔵) and donors (🔴) are available in

• the major groove (AT, TA, GC, CG can be discriminated)

• the minor groove (AT/TA vs. GC/CG can be discriminated

Amino acid side chains form hydrogen bonds with bases in double helical DNA

Sequence-specific DNA recognition is key to carrying out the steps in information transfer

Asn

• Gln

• Glu

• Lys

• Arg

DNA binding proteins

• Generally bind DNA in the Major Groove

• a-helix fits nicely into the wide major groove

Certain DNA Binding Motifs are common (5)

1. Helix-Turn-Helix (HTH)

2. Zinc Finger

3. Homeodomain

4. Leucine Zipper

5. Basic Helix-Loop-Helix (BHLH)

DNA Binding Motifs: Leucine Zipper

1. Lys and Arg residues ➡DNA binding + in the major groove

   1. Leu residues stabilize dimerization

DNA binding motifs: homeodomain

The homeodomain is a widely conserved DNA binding motif of ~ 60 amino acids

DNA binding motifs: helix-turn-helix

Recognition Helix is positioned in the Major Groove

Helix-turn-helix domains in the lac repressor protein

DNA binding motifs: zinc finger

Coordinated Zn2+ ion (4 Cys or 2 Cys, 2 His)

DNA binding motifs: helix-loop-helix

amphipathic helices mediate dimerization

Basic residues mediate DNA binding

DNA must be compacted to fit in cells

length details?

The length of T4 DNA is several hundred times the size of the viral particle.

If you add up the length of the DNA in all cells of an average human

(~1014 cells) the total DNA length is ~ 2 x 1011 km.

The distance between the earth and the sun is 1.5 x 108 km.

Supercoiling does 2 things:

Supercoiling compacts DNA

Supercoiling relieves strain

Polymerases separate the strands and introduce strain

Supercoiling is an intrinsic property of DNA tertiary structure.

• It occurs in all cellular DNAs and is highly regulated by each cell.

Most cellular DNA is underwound: Supercoiling _________ __________

Underwinding facilitates strand separation

DNA underwinding is defined by the topological linking number, Lk

Separation of two strands of a double-stranded circular DNA.

In DNA, Lk specifies the number of helical turns in a closed circular DNA

Lk = #bps ÷ bps per turn

Relaxed DNA:

2100 bp DNA / (10.5 bp/turn)

Lk = 2100 ÷ 10.5

Lk = 200

Superhelical Density

Negative and positive supercoils

Two forms of a circular DNA that differ only in a topological property such as linking number are referred to as _________

Topoisomers.

Underwind by 2 turns ➡∆Lk = –2

• So, Lk = 200 – 2 = 198

Overwind by 2 turns ➡∆Lk = +2

• So, Lk = 200 + 2 = 202

topoisomerase

Type I • changes Lk in increments of 1 • cleaves one strand of duplex DNA • can relax positive and negative supercoils • example: Topoisomerase I

Type II • changes Lk in increments of 2 • cleaves both strands of DNA • can relax positive and negative supercoils • can introduce negative supercoils (prokaryotes only) • hydrolyzes ATP • examples: DNA gyrase, Topoisomerase II

Type I Topoisomerases

• change Lk in increments of 1

• cleave one strand of duplex DNA

• can remove negative supercoils

Topoisomerase I steps

Topoisomerase II reactions

Plectonemic and solenoidal supercoiling

Plectonemic supercoiling does not produce sufficient compaction to package DNA in the cell.

DNA is packaged into nucleosomes

Histone proteins act to package the DNA