DNA Replication & Genetics

Central Dogma

DNA → RNA → Protein

DNA Replication Direction

5' → 3'

DNA Polymerase III

Main replicative enzyme in E. coli

DNA Polymerase I

Removes RNA primers and replaces with DNA

DNA Polymerase II

DNA repair and lesion bypass

DNA Polymerase IV

Error-prone repair polymerase (SOS)

Helicase

Unwinds DNA using ATP

Primase

Synthesizes RNA primers

Okazaki Fragments

Short DNA segments on lagging strand

β Sliding Clamp

Increases DNA polymerase processivity

Exonuclease Activity

Proofreads DNA during replication

DNA Supercoiling

Compacts bacterial DNA

DNA Gyrase

Introduces negative supercoils

Topoisomerase

Relieves DNA strain

Genome

Complete set of DNA in an organism

Histones

Proteins that wrap DNA in eukaryotes

Histone Acetylation

Increases DNA accessibility

Transposons

Mobile DNA elements

Transposase

Enzyme that moves transposons

Barbara McClintock

Discovered transposons

Replisome

Protein complex that carries out DNA replication.

Sense Strand

Same sequence as mRNA except T → U.

Antisense Strand

Template strand used in transcription.

E. coli DNA Polymerases

E. coli has 5 DNA polymerases (I-V).

DNA Polymerase Structure

Hand-like with palm, fingers, and thumb domains.

Palm Domain

Catalyzes bond formation.

Finger Domain

Binds incoming nucleotides.

Thumb Domain

Increases processivity.

Exonuclease Fidelity Improvement

Improves accuracy ~1000x.

Clamp Loader

Loads β sliding clamp onto DNA.

Pol III Subunits

α (polymerase), ε (proofreading), θ (stabilizer).

SSB Proteins

Stabilize single-stranded DNA.

DNA Polymerization Mechanism

3'-OH attacks α-phosphate of dNTP.

Pyrophosphate (PPi)

Released during DNA synthesis.

PPi Hydrolysis

Drives DNA replication forward.

Metal Ions in Replication

Mg2+ ions assist catalysis.

oriC Length

~245 base pairs.

AT-rich Regions

Easier to unwind for replication.

Tus Protein

Stops replication at termination sites.

E. coli Genome Size

~4.6 million base pairs.

B. subtilis Genome Size

~4.3 million base pairs.

Protein Coding Percentage (E. coli)

~70-80%.