Dna replication

DNA REPLICATION

Meselson and Stahl (1958)

1958: Matthew Meselson & Frank Stahl’s Experiment

Semiconservative model of DNA replication

1958: Matthew Meselson & Frank Stahl’s Experiment

Equilibrium density gradient centrifugation

Arthur Kornberg

Worked with E. coli. Discovered the DNA POLYMERASE nd describe mechanisms of DNA synthesis.

Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)

2. DNA template

3. DNA polymerase (Kornberg enzyme)

4. Mg 2+ (optimizes DNA polymerase activity)

1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)

Model of DNA replication

Model of DNA replication

The Chemistry of DNA replication

DNA HelicaseDNA double helix are tightly coupled. High temperature is needed to break them (95oC)

DNA Synthesis by DNA polymerase

DNA PolymeraseNucleotide polymerizing enzyme, first discovered in 1957

DNA replication Fork

DNA Binding ProteinSSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

SSB Proteins

DNA

DNA Clamping Protein

Cycle of DNA Polymerase/Clamping Protein loading and unloadingAt the lagging strand..

Machinery for DNA replication

A Moving Replication

Structure of the Moving Complex

DNA winding

Topoisomerase

Opening the dsDNA will create supercoil ahead of replication forks.

The supercoil constraint needs to be released by topoisomerases.

The interconversion of topoisomers of dsDNA is catalyzed by a topoisomerase in a three-step process: Cleavage of one or both strands of DNAPassage of a segment of DNA through this

breakResealing of the DNA break

It is named gyrase in prokaryotes.

It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds.

Topoisomerase II (topo II)

Also called -protein in prokaryotes.

It cuts a phosphoester bond on one DNA strand, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut.

Topoisomerase I (topo I)

What about the ends (or telomeres) of linear chromosomes?

DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. this gap is not filled, chromosomes would become shorter each round of replication!

Solution:

1. Eukaryotes have tandemly repeated sequences at the ends of their chromosomes.

2. Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of of new repeats.

3. Compensates by lengthening the chromosome.

4. Absence or mutation of telomerase activity results in chromosome shortening and limited cell division.

Telomere replication

DNA Proofreading

Site-directed mismatch repair in eucaryotesIn DNAs are usually methylated on A while newly synthesized ones are not. So Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

Model of DNA replication

Model of DNA replication