102 handout 1122 - MCB102 Handout 11/22 This handout covers...

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MCB102 Handout 11/22 This handout covers lectures 5-8. You should look over the reader, your class notes, and the assigned book pages for clarification. Unless otherwise noted, the processes occur in the bacterium E. coli . REPLICATION (continued) INITIATION 1. Dam methylase methylates adenines in the sequence GATC, which occurs many times at the origin of replication (called oriC in E. coli ). This signals that it’s time to replicate. 2. DnaA and HU, a histone-like protein, bind to the many DnaA box sequences present at the origin. DNA is wrapped around the proteins, making the DNA positively supercoiled. 3. DnaA recognizes AT-rich repeats nearby and, along with pre-priming proteins, denatures them (separates the DNA strands). This process requires ATP but is facilitated by positive supercoiling. 4. DnaC helps the helicase DnaB bind, and DnaB further unwinds the DNA. 5. DNA gyrase (topoisomerase II) relieves the strain created by the unwinding of DNA and the separated strands are bound by single-stranded DNA binding protein (SSB). 6. DnaG (primase) synthesizes the first RNA primers. ELONGATION (See lecture 5 pages 74-75 or book page 961- the thick black arrow indicates the direction the template DNA moving through the polymerase since it is the DNA, not the polymerase, that moves) -Replication is bidirectional ; unwinding at the origin creates 2 replication forks. A DNA polymerase III dimer (two attached polymerases) binds each fork and proceed in opposite directions from the origin. - E. coli ’s circular chromosome has one origin. Human linear chromosomes each have many. -The DNA polymerase III dimer and synthesizes both strands of DNA in the 5’ to 3’ direction. -It is held onto the DNA by a protein called the β clamp that encircles the DNA. . -DNA polymerase III adds dNTPs to the leading strand primer continuously. -DNA polymerase I removes RNA primers and fills in the gap with DNA. -DNA ligase repairs the nick (single-strand break) that remains after DNA polymerase I action. -The polymerases involved in human DNA replication are called δ (delta) and ε (epsilon). Steps unique to lagging strand synthesis 1. The primer of the previous Okazaki fragment (old primer) approaches the core, which stimulates primase to make a new primer and the clamp loading complex to load a new β clamp. 2. The polymerase runs into the old primer, stops synthesis and dissociates from the old clamp. 3. The polymerase “jumps back” and binds the new clamp at the site of the new primer 4. The polymerase begins elongating again. REPLICATION OF LINEAR CHROMOSOME ENDS (HUMANS) -Removal of the last lagging strand primer leaves a ~100 nucleotide gap at the 5’ end. Unless this is fixed, chromosomes would shorten after each round of replication. -Telomerase uses a bound RNA template to add hundreds of copies of a repeating sequence (AGGGTT in humans) to the 3’ end. Chromosome ends with this sequence are called telomeres.
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