Molecular and Cellular Basis of Life: DNA Replication

These flashcards cover the fundamental rules, experimental evidence, key enzymes, and stages of DNA replication in E. coli as presented in the lecture transcript.

Semiconservative Replication

A mechanism of DNA replication where each new DNA molecule consists of one old parent strand and one new daughter strand.

Meselson and Stahl Experiment

A 1958 study using $^{15}\text{N}$ and $^{14}\text{N}$ isotopes and CsCl density gradient ultracentrifugation to prove that DNA replication is semiconservative.

Cairns’ Experiments

Research where DNA was radiolabeled with $^3\text{H}$ (tritium) and visualized using photographic emulsion to show that bacterial DNA replication is bidirectional and involves two replication forks.

Inman’s Experiments

A denaturation mapping technique showing that DNA is selectively denatured at sequences rich in $A=T$ base pairs, generating reproducible single-stranded bubbles at unique origins.

$5' \rightarrow 3'$ Direction

The universal direction of DNA synthesis, where new nucleotides are always added to the $3'\text{-OH}$ end of the growing strand.

DNA Polymerase III

The principal replication polymerase in $E.\,coli$ characterized by high processivity and a complex structure containing 10 types of subunits.

DNA Polymerase I

A polymerase in $E.\,coli$ with low processivity that possesses both $3' \rightarrow 5'$ and $5' \rightarrow 3'$ exonuclease activity; it is primarily involved in error correction and the removal of RNA primers.

Replisome

A large protein complex consisting of over 20 different enzymes and proteins, such as helicases, primases, and ligases, required for DNA replication in $E.\,coli$.

oriC

The unique origin site in $E.\,coli$ that is $245\,bp$ long and contains five repeats of a $9\text{-bp}$ sequence ($R$ sites) and an $A=T$-rich DNA unwinding element ($DUE$).

DnaA protein

An initiator protein that recognizes the ori sequence and opens the DNA duplex at specific sites by inducing positive supercoiling and strain.

DnaB protein (Helicase)

A hexameric protein that unwinds DNA by migrating along single-stranded DNA in the $5' \rightarrow 3'$ direction.

DNA Gyrase (DNA Topoisomerase II)

An enzyme that relieves the torsional strain generated by the unwinding of the DNA double helix.

Primase (DnaG protein)

A specialized enzyme that synthesizes short RNA primers to provide a starting point for DNA polymerases.

Okazaki Fragment

Short segments of DNA synthesized discontinuously on the lagging strand during replication.

$\beta$ clamp (Sliding Clamp)

A dimer of subunits within DNA Polymerase III that encircles DNA and significantly increases the processivity of the polymerase complex.

DNA Ligase

The enzyme responsible for sealing "nicks" in the DNA backbone by joining fragments together after RNA primers are removed and replaced with DNA.

Ter sites

$20\text{-bp}$ sequences where replication forks meet to terminate replication in $E.\,coli$, acting as a functional "trap."

Tus (Terminus Utilization Sequence)

A protein that binds to $Ter$ sites and arrests replication fork progression by blocking the action of the $DnaB$ helicase.

Dam methylase

An enzyme that methylates $(5')\text{GATC}$ sequences at the $oriC$ site, playing a role in the initiation of replication.

SSB (Single-stranded DNA-binding protein)

Proteins that bind to and stabilize separated single DNA strands to prevent them from re-annealing during replication.