biol1406 chapter 14

griffith’s experiment

showed transformation in bacteria; harmless R strain became virulent when mixed with heat-killed S strain

transformation (griffith)

genetic material from dead S cells entered live R cells, converting them into virulent bacteria

avery, macleod, mccarty

identified DNA as the transforming principle; only DNA from S strain transformed R strain

hershey-chase experiment

used bacteriophages labeled with radioactive DNA or protein; proved DNA is injected into bacteria and is the genetic material

DNA nucleotide components

deoxyribose sugar, phosphate group, nitrogenous base (A, T, C, G)

phosphodiester bond

covalent bond linking nucleotides between the phosphate of one and the 3’ OH of the next

chargaff’s rules

A=T, G=C; purines pair with pyrimidines

roalind franklin contribution

x-ray diffraction images revealed DNA is helical, 2 nm diamter, 3.4 nm per turn

watson and crick

built the double-helix model using chargaff’s rule + franklin’s data

base pairing rules

A-T (2 hydrogen bonds) and G-C (3 hydrogen bonds)

antiparallel strands

DNA strands run in opposite directions: one 5’ → 3’, the other 3’ → 5’

Conservative model

Original DNA stays intact; all new DNA is in the daughter molecule

semiconservative model

Each daughter DNA has one old strand and one new strand

Dispersive model

Old and new DNA are mixed in every strand

Meselson–Stahl experiment result

Confirmed the semiconservative model using ¹⁵N/¹⁴N density labeling

Requirements for replication

Template DNA, enzymes, nucleoside triphosphates (dNTPs)

initiation

Replication begins at origins; DNA unwinds and primers are laid down

elongation

DNA polymerase synthesizes new DNA strands

termination

Replication ends; daughter molecules separate

DNA polymerase direction

Adds nucleotides only to the 3′ end (synthesizes 5′→3′)

Primer requirement

DNA polymerase needs an RNA primer to begin replication

Energy for DNA synthesis

Comes from cleavage of phosphates off dNTPs (release of pyrophosphate)

Origin of replication (prokaryotes)

Single origin (OriC) on circular chromosome

Bidirectional replication

Two replication forks move in opposite directions around chromosome

DNA polymerase III

Main replication enzyme

DNA polymerase I

Removes RNA primers and replaces them with DNA

DNA polymerase II

DNA repair enzyme

Helicase

Unwinds DNA helix using ATP

Single-strand-binding proteins (SSBs)

Keep DNA strands separated

Topoisomerase / DNA gyrase

Relieves torsional strain and prevents supercoiling

Primase

Makes RNA primers for DNA polymerase to extend

DNA ligase

Joins Okazaki fragments on the lagging strand

Leading strand

Synthesized continuously toward the replication fork

Lagging strand

synthesized discontinuously away from the fork (Okazaki fragments)

Okazaki fragments

Short DNA pieces synthesized on the lagging strand

Replisome

Large replication complex containing helicase, primase, and two DNA polymerase III enzymes

Multiple origins

Eukaryotic chromosomes have many origins to speed replication

Chromatin challenge

DNA must be unpacked and repacked around histones during replication

Primer removal (eukaryotes)

RNase H + DNA polymerase δ

Main eukaryotic polymerases

Pol α (primer synthesis), Pol δ & ε (replication)

Telomeres

Protect ends of chromosomes; shorten with each division unless extended by telomerase

Telomerase

Enzyme that extends telomeres; active in stem cells and cancer cells

Mutagens

Agents that increase mutation rate (UV radiation, chemicals)

Specific repair

Targets and fixes one type of DNA damage (e.g., mismatch repair)

Mismatch repair (MMR)

Corrects incorrectly paired bases after replication by distinguishing old vs new strand

Excision repair

Damaged DNA region is cut out and replaced using the intact strand as template