• Millions of nucleotides in length • Replication must occur very quickly and with minimal errors • Watson and Crick’s 3-D model of DNA gave scientists a place to begin DNA Replication is Semi-Conservative
• Semi-conservative replication involves
separating the two parent strands and building a new, complementary replacement strand for each • New molecule consists of one parent strand and one new strand • Conservative replication on the other hand would mean that the original parent strand would stay together • Meselson and Stahl (1958) used isotopes and e. coli bacteria to label parent DNA strands before replication (Pg. 282. Fig. 1) • Each single strand of parent DNA has been used as a template for a new complementary DNA strand DNA Replication: The Process
• There are three main
steps to DNA replication: 1. The parental strands of DNA separate 2. The complementary DNA strands are assembled 3. The new strands are proofread and repaired Step 1: Strand Separation
• The DNA must first be unwound from
each other • Specific nucleotide sequences act as replication origins (starting points) • There can be many different replication origins per strand due to the length • Enzyme called Helicase binds to the replication origins and begins to unwind strands by breaking the hydrogen bonds between the base pairs • Separation of strands creates a Y-shaped replication fork Step 1: Strand Separation • A class of enzymes called topoisomerases relieves the tension caused by the unwinding of parent DNA • They cleave one or two of the DNA strands, allowing them to untwist, and then rejoin the stands • Single strand binding proteins (SSBs) prevent annealing (strands coming back together before they should) • Helicase can separate in both directions from replication origin creating replication bubbles of newly formed DNA • Numerous replication bubbles allows for new DNA to be formed at 50 base pairs per second Step 2: Building Complementary Strands • New nucleotides are joined by a group of enzymes called DNA polymerases • DNA polymerases add nucleotides to the 3’ end of a new developing strand reading the template strand 5’ – 3’ • Therefore, new strand can only be assembled in the 5’ to 3’ direction • DNA builds new strand using nucleoside triphosphates (building block and energy source) • Energy to drive the DNA synthesis is provided by hydrolysis of phosphate groups Step 2: Building Complementary Strands
• RNA primase builds a small
complementary RNA segment at the beginning of the replication fork • DNA polymerase III begins adding DNA nucleotides to the RNA primer in the 3’ – 5’ direction • Therefore, two new strands will begin to be assembled in opposite directions • As the replication fork continues to open, The DNA polymerase III building the strand towards the fork can continuously add nucleotides • This is called the leading strand (new strand heading towards replication fork) Step 2: Building Complementary Strands
• On the other template strand DNA polymerase is
moving away from the replication fork (Lagging strand) • When enough fork has opened RNA primase attaches RNA primer to the template (parent) strand • Therefore, the lagging strand gets built in sections called Okazaki Fragments • Lagging strand is discontinuous (it needs multiple RNA primers and Okazaki fragments to be completed • Another polymerase, DNA polymerase I removes RNA nucleotides and replaces with DNA • DNA ligase catalyzes the final linkage between the DNA segment and the Okazaki fragment Step 3: Dealing with Errors during DNA
• DNA polymerase enzymes proofread and
correct errors as they build new strands • Average of 1 error in every million base pairs • Once, done, DNA polymerase I and II read the new strands for errors • DNA polymerase fixes the left over errors and DNA ligase fuses the strand together • These are constantly working to correct damage to DNA from environmental factors (chemicals and radiation) DNA Replication in Prokaryotes and Eukaryotes • Most DNA knowledge has come from bacteria studies • Process is very similar in eukaryotes • Prokaryote genomes can be different shape (circular) or smaller than eukaryotes • Only one replication origin and bubble in eukaryotes • Understanding DNA replication could have significant implications for growth, aging, cloning, tissue regeneration, cancer, and biotechnology