DNA The Molecule of Life: Replication

Replication: Why? When cells replicate, each new cell needs it’s own copy of DNA. Where? Nucleus in Eukaryotes. Cytosol in Prokaryotes

Replication: When? S phase of cell cycle (S for synthesis, during interphase) What? Many proteins involved as enzymes: major one is DNA Polymerase How?

Replication (def’ns) Using one side of the DNA strand as a TEMPLATE to make a complementary side.

Replication - (def’ns-cont’d) Template – parental strand Complementary copy – daughter strand Both sides of the DNA double strand do this. End result – two identical double helixes (helices).

DNA Replication is Semiconservative Each double helix has one parent strand that is conserved and one daughter strand that is new The new strand forms by base-pairing that is complementary to the parent strand. (A-T and C-G) Meselson and Stahl Experiment BIOLOGY.htm

Semiconservative:

Replication: Here’s How How? Enzymes! 5’  3’ directionality Starts with RNA primer Leading Strand Lagging Strand Okasaki Fragments Sequence determined by base pairing DNA Replication Animation

Replication: Prerequisites Parent DNA needs to unwind and “unzip” The hydrogen bonds between the paired bases break. RECALL hydrogen bonds are not really bonds, they are forces between atoms of H and either N or O (for DNA and RNA). Weaker than covalent bonds in rungs Phosphate/sugar bond Nucleotide bases are always present in the nucleus

Replication Enzymes 1. Helicase: the enzyme required to unwind and split the H-bonds in the DNA double helix – exposing the bases. 2. Primase is the enzyme that lays down the RNA primer (establishes starting point) Needs a “primer” or a place to start from—they cannot pick up the first two required nucleotides unless they have the RNA primer (primase).

DNA Polymerase: 3. DNA Polymerase III recognizes the exposed bases and matches them up with free, complementary nucleotides (H bonds). 4. The enzyme then bonds the sugars and phosphates together to form the backbone of the new strand

Proofreading 5. Makes only one mistake every 10^8 or 10^11 bases it adds. Has proof-reading abilities! A single strand of DNA has about 10^7 – 10^11 base pairs DNA replication occurs fairly quickly; as many as 4000 nucleotides per second are replicated. This helps explain why bacterial cells, under ideal conditions, can reproduce in 20 minutes.

Proofreading (cont’d) 6. DNA polymerase I removes the RNA primers when done and fills in the RNA gaps with DNA. DNA ligase seals any nicks in the DNA by linking up the deoxyribose to the phosphate to seal the new DNA strand together. There are a few others, but this is enough for now.

Enzyme Summary

Deoxy vs ribose sugar

5’ to 3’ Direction

5’ to 3’ Animation

Replication is 5’-3’ Replication is always 5’ to 3’. But this means one strand is made in one long segment but the other ends up in fragments… DNA-replication-an-existing-DNA-molecule-is-separated

One strand: Continuous One strand of DNA is synthesized continuously from 5’ to 3’ end. This strand is called the leading strand It is called this because it requires only one primer Replication proceeds in the same direction/towards as the replication fork in the DNA strand At the end of the strand, a TELOMERE is added by another enzyme. Telomere: none-coding redundant sequences.

Other: Discontinuous The other strand of DNA is synthesized discontinuously – in pieces – because it also needs to be made 5’ to 3. This strand is called the lagging strand. The many small fragments are called Okazaki fragments

Other: Discontinuous It requires multiple primers because it replications AWAY from the replication fork As a fork opens up, a RNA primer attaches, DNA polymerase adds bases 5’ to 3’ until it reaches the “other” RNA primer. This process continues as the fork opens up until the end of the DNA. At the end, another enzyme adds a telomere.

DNA polymerase assembles new complementary strands

SUMMARY and demo How? Enzymes make it happen correctly. There are many 5’  3’ directionality for both strands Starts with RNA primer for DNA polymerase to attach Leading Strand is continuous Lagging Strand is discontinuous Okasaki Fragments are connected by polymerase I Sequence determined by complementary basepairing enzyme roles Replication animation-slow Animation-johnkyrk Animation-fast

Sources of info, Assignments Good sources of info: Your textbook! Chapter 11 pg Especially pg Copying DNA Fig 11.5 Assignment Worksheets Text pg. 287 Q 1-5 ? The internet has a LOT of good animations you could view. Google DNA replication animations.