Chapter 10 | DNA Structure and Analysis

Phosphodiester Bond Formation

Two nucleotides join through dehydration synthesis, linking the phosphate of one nucleotide to the 3’ carbon of another sugar

Ligase function in NHEJ

DNA ligase regenerates phosphodiester bonds by connecting a monophosphate to a base using ATP hydrolysis (a nonspontaneous, exergonic reaction)

Energy of phosphodiester bond formation

Formation of the bond between the OH of the phosphate and the 4' carbon is endergonic (requires energy input)

Sugar in a nucleotide

A 5-carbon sugar where:

• 1′ carbon attaches to base via a glycosidic linkage,

• 2′ carbon has OH (ribose) or H (deoxyribose),

• 3′ and 5′ carbons allow DNA chain formation,

• A missing 4′ OH prevents phosphodiester bonding

Chargaff’s findings

• A = T, G = C

• A + T ≠ G + C

• A + G = C + T → purines = pyrimidines

• Ratios consistent across all organisms

DNA double helix structure

DNA forms a right-handed double helix with a phosphate backbone outside and bases inside held by hydrogen bonds

DNA backbone

Negatively charged and hydrophilic, facing the aqueous environment

Base orientation

Bases are hydrophobic and face inward, pairing via hydrogen bonds (A–T, G–C)

Antiparallel strands

Two DNA strands run in opposite directions (5′→3′ vs 3′→5′)

Helix dimensions

• 1 base pair = 3.4 Å apart

• 10 base pairs per turn (~34 Å)

• Diameter = 20 Å constant due to purine–pyrimidine pairing

DNA grooves

Alternating major and minor grooves where proteins interact noncovalently (H-bonds, ionic, van der Waals); major groove provides more information

Base pairing rule

Purines (A, G) pair with pyrimidines (T, C) to maintain uniform diameter

A-T pairing

A (purine) pairs with T (pyrimidine) via 2 hydrogen bonds

G-C pairing

G (purine) pairs with C (pyrimidine) via 3 hydrogen bonds, making the pair more stable

Hyperchromic shift

Increase in UV absorbance (260 nm) as DNA transitions from double-stranded to single-stranded during heating

Cause of increased absorbance

In single-stranded DNA, nitrogenous bases are more exposed to UV light, increasing absorption

DNA melting temperature (T_m)

Temperature where half of DNA is denatured (≈77 °C for typical DNA)

Base composition and melting point

GC-rich DNA requires higher temperature (≈83 °C) to melt due to 3 H-bonds per base pair, compared to 2 in AT pairs