DNA replication

DNA replication

• is semiconservative

• Researchers proposed two possible mechanisms of DNA replication

1. conservative replication: the two parental strands act as templates for replication but then recombine afterwards

2. semiconservative replication: the two parental strands act as templates for replication and remain separated from each other, being incorporated into two new molecules

Meselson-Stahl experiment (1958)

• grew E.coli bacteria in a medium contains “heavy: nitrogen isotope (15N) for any generations

• transferred those bacteria to a medium with normal, “light” nitrogen (14N)

• allowed only one or two rounds of replication

• DNA isolated and centrifuged in a mixture that produces a density gradient - DNA would migrate to a level in the tube with similar density

• Conclusion: DNA replicates semi-conservatively

The process of replication

• Replication begins at special sites called origins of replication

• Replication spreads in both directions along the DNA from these central initiation points

Prokaryotes vs eukaryotes

• A bacterial DNA molecule only contains one replication origin while a eukaryotic chromosome has 100s

• Enzymes involved in eukaryotes are more complex than outlined

Helicase

• Binds to replication origin and unwinds the DNA by breaking H-bonds between complementary bases

• the two strand will form a Y shaped structure called the replication fork

Topoisomerase

• DNA gyrase

• relieves the tension from unwinding of the double helix by creating nicks in the DNA so the strand can rotate and then resealing the nick

Single-stranded binding proteins

• stabilize the wound strans and prevent them from reannealing

RNA Primase

• builds short complementary segments of RNA (10-60) nucleotides on the template strands

• Each segment is called a primer

• Required because DNA polymerase iii can only add new nucleotides to existing ones

DNA polymerase iii

• adds nucleoside triphosphates starting at the end of the RNA primers

• Nucleotides can only be added in the 5’ → 3’ direction (meaning adding to the 3’ end of the growing strand)

Nucleoside Triphosphates

• have 3 phosphates which store energy as in ATP

• when 2 phosphates (pyrophosphate) hydrolyze, this provides the energy needed by DNA pol iii to catalyze the synthesis reaction to add a new nucleotide to the strand

Leading strand

• This strand is built in the same direction as the replication fork

• It is built continuously in one long segment and requires only one primer

Lagging Strand

• This strand is built in the opposite direction as the replication fork

• it is synthesized discontinuously in short fragments called Okazaki fragments

• Each fragment must be started by an RNA primer before it can be extended by polymerase

DNA polymerase i

• removes the RNA nucleotides from the primers and replaces them with the appropriate DNA nucleotides

DNA Ligase

• joins unattached sections of DNA by forming phosphodiester bonds between them

• Required many times on the lagging strand but only once on the leading strand

Exonucleases

• DNA polymerase iii sometimes adds the wrong nucleotide (once in 1 million b.p.)

• DNA polymerase II and DNA polymerase I have exonuclease functions that allow them to remove mismatched DNA nucleotides and replace them with the correct ones