Molecular Genetics 2: DNA Replication

WHAT IS DNA REPLICATION? The process of making two identical DNA molecules from an original, parental DNA molecule Occurs during the “S-phase” of Interphase We need to copy our DNA so that when our cell divides into 2 cells, each cell will have a complete copy of the DNA. There were three proposed models for DNA Replication –Conservative (replication by forming 2 new daughter strands and joining them to create a brand new DNA) –Semi-Conservative (each new DNA contains one old and one new strand) –Dispersive (parental DNA is broken into fragments and then copied so the new DNA contains pieces of old and new randomly) Meselson and Stahl designed an experiment using isotopes to figure out that the semi-conservative model was correct.

Semi-Conservative Replication Phase 1: Initiation Must start at a specific nucleotide sequence called the origin of replication Several initiator proteins will bind onto the DNA and start to unwind it. –One group of proteins are called the helicases. They cleave the hydrogen bonds that link the complementary base pairs –Single-strand-binding proteins help to stabilize the unwound single strands –Topoisomerase II enzyme helps to relieve strain on the double helix sections ahead of the replication area This initiation process creates an unwound oval shape area called the replication bubble with two Y-shaped regions at either end called the replication forks As replication continues, the replication forks move along the DNA in opposite directions.

DNA Replication Phase 2: Elongation This phase makes new DNA strands by joining nucleotides together on the original complementary DNA strand DNA polymerase III is the enzyme that catalyzes the addition of new nucleotides one at a time DNA polymerase III can only work by attaching the new nucleotides to the free 3’ -OH end of a pre-existing end. DNA polymerase III works in the 5’ to 3’ direction only.

Elongation Continued Because when the DNA is unwound, we don’t have any free 3’ -OH’s to start to use a primer. The primer is actually made of RNA and is laid down by the enzyme primase Leading Strand: –Travels in the 5’ to 3’ direction with the direction of the replication fork –Only needs one primer to start the process Lagging Strand: –Travels in the 5’ to 3’ direction opposite the replication fork –Each primer is laid down as the replication bubble moves along –Must lay down many primers –After each primer is added the DNA polymerase III can add the DNA to the free 3’ end. –Because this happens in chunks, it doesn’t create one long chain, but smaller fragments called Okazaki fragments

Elongation Continued DNA polymerase I then comes in to remove all the RNA primers and fills in the spaces with DNA The Okazaki fragments are then joined together by the enzyme called DNA ligase

DNA Replication Phase 3: Termination As the replication fork progresses along the replicating DNA, the newly formed strands will rewind automatically into their stable double helix shape. The two new DNA molecules separate from each other and the “replication machine” is dismantled.

Correcting the Errors One error that can occur is the miss-pairing between a new nucleotide and a nucleotide on the template strand. (EG a T might be paired with a G instead of an A) Another mistake can be due to strand slippage which causes either additions or deletions of nucleotides Thankfully we have DNA polymerase II, which acts as a proofreader (with the help of DNA polymerase I) As they come across the error, the DNA polymerases take out the wrong nitrogenous base and add the correct one. Mismatch repair is another form of proofreading where the miss-pairing of bases causes deformities that are recognized by a group of enzymes that will remove the incorrect base. Any errors that are not caught by the DNA polymerases or mismatch repair are then considered a mutation.

DNA Replication in Prokaryotes vs Eukaryotes Both –Require origins of replication –Have elongation in the 5’ to 3’ direction –Continuous synthesis of leading strand –Discontinuous synthesis of the lagging strand –Need a primer for Okazaki fragments of the lagging strand –Use DNA polymerase enzymes Differences: –Rate is faster in prokaryotes –DNA polymerase enzymes are slightly different –Prokaryotes will have a single origin of replication while eukaryotes have thousands of origins. –Eukaryotes have telomeres (repetitive sections of DNA at the end of each chromosome that helps protect from a loss of genetic information) Telomerase enzymes can help build back up the telomeres that may have been lost during replication.

ReviewReview Page 229 Questions 1, 3-7, 9 and 10.