DNA Replication Structure of DNA was discovered using model building. Chemical components of DNA. Chargoff’s Rule Covalent bond length and angle. Critical size measurement from X-Ray diffraction of DNA. Thermodynamics Non-Covalent interactions (Base pairs)

Chemical component of Single Stranded DNA Backbone composed of deoxyribose sugars connected by phosphodiester bonds. 4 nitrogenous bases (T, A, G, C) Polar (5’ end and 3’ end are different)

Ester/Phosphodiester Linkage

Chargoff’s Rule The amount of G, C, A, T varies from species to species. In all species – Amount of A = Amount of T – Amount of G = Amount of C

X-Ray Crystallography X-rays are high energy light (photons) Pure solution of molecules is crystallized, so the molecules are in an ordered array, each one oriented in the same way. Regularly spaced atoms in the crystal deflect x- rays in an ordered pattern. The x-rays strike and darken a piece of film (spots). The arrangement of spots is a reflection of the arrangement of the atoms in the crystal.

X-Ray Chrystallography

Structure of DNA DNA is a double helix Negatively charged, hydrophilic sugar- phosphate backbone. Hydrophobic base pairs. Strands are anti-parallel so that base pairs can be formed. T with A G with C

Major and Minor Grooves DNA has 2 helical strands. The spaces between the strands form 2 helical grooves. Due to the fact that the strands are not exactly opposite to each other, the grooves are not the same.

DNA Rotating Lqc

DNA Polymerases Synthesize Complementary strands of DNA The 2 strands of DNA unwind. Each DNA strand serves as a template for new strand synthesis. Base pair rules followed to direct synthesis of the new strand of DNA The sequence of a newly synthesized strand is complementary to its template strand.

Deoxynucleotide Triphosphate (dXTP) Deoxyribose sugars are numbered 1’ to 5’. Groups attached to 5’ and 3’ carbons become sugar-phosphate backbone. 2’ carbon has hydrogen. Innermost phosphate part becomes part of sugar- phospate backbone. Outer 2 phosphates (pyrophosphate) cleaved off during DNA synthesis. Important in providing energy to create bond.

dXTP

Adding a nucleotide to the 3’ end 5’ to 3’ synthesis Free electron pair from 3’ hydroxyl (OH) attacks the slightly positively charged innermost phosphate of dXTP forming a covalent bond. Pyrophosphate is the leaving group, energy is released. Energy for making phosphodiester bond is built into dXTP.

DNA Replication is bi-directional

DNA Polymerase III Accounts for 99% of all replication activity. Enzyme complex for most of DNA synthesis. Simultaneously copies both template strands. Can only synthesize DNA in the 5’ to 3’ direction. Cannot initiate DNA synthesis. Can only elongate from the 3’ end of a pre-existing nucleic acid dXTP’s per second.

Error rate of DNA Poly III DNA Poly III inserts the wrong base one out of 10 7 times. 10 million. This is very good, but not good enough to replicate large genomes like humans, with over 10 9 base pairs. Results in 100 mistakes every time DNA divides. Lethal. Proofreading greatly reduces the error rate.

Proofreading by DNA Poly III Wrong base does not fit. It wobbles. From 3’ to 5’, hydrolyzes last phosphodiester bond. 5’ to 3’ synthesis. Hydrolyzing the last 3’ nucleotide.

Proofreading requires 5’ to 3’ synthesis

Hydrolyzing the last 3’ nucleotide Needs to be done in proof reading phase.

DNA Polymerase cannot initiate synthesis To begin DNA replication/synthesis we need a primer. That initates elongation of DNA strand Primer is created by an enzyme called RNA Primase. Can only make 5-10 bases long. DNA Polymerase III uses open 3’ hydroxyl of RNA primer to attach next DNA nucleotide.

DNA Polymerase III

DNA Looping allows for replication in same direction

Primase makes a short RNA Primer for DNA Synthesis initiation Primase initiates primer synthesis. DNA Polymerase III synthesizes DNA starting from the 3’ OH of the primer.

DNA Polymerase I removes the RNA primer and synthesizes DNA in its place DNA Polymerase I is a monomer, works on one strand, has two active sites. 5’ to 3’ hydrolysis activity. 5’ to 3’ DNA synthesis activity.