Biol 2334 Molecular Biology Chapter 5 Worksheet Replication & Repair

1. When read in the same direction (5 3), the sequence of nucleotides in a newly synthesized DNA strand is the same as in the parental strand used as the template for its synthesis. True or False and why? False (in most cases). The template strand is the complement of the newly synthesized strand. Thus, they are not identical, they are complements of one another. If the parental strand is a perfect palindrome, then the newly synthesized strand will read the same 5 3 as the parent strand. Eg. Template strand: 5-ATGCAAGTC-3, New strand: 5-GACTTGCAT-3 Palindrome example: Template 5- ATGCGCAT-3, New: 5-ATGCGCAT-3 2. Okazaki fragments are typically longer in prokaryotes than eukaryotes. What is one possible reason for this difference in length? Activity of primase if primers are layed down more often, there will be shorter fragments. Also processivity of polymerase more processive would require fewer primers and result in longer fragments. Presence of nucleosomes in euks may also affect how long okazaki fragments are. 3. Draw and label the leading and lagging strand of the replication fork below. Be sure to label the 5 and 3 ends of both template and synthesized strands. (Yes, there are two possible ways to label this you pick!).
3 5 5 3 3 5

Leading strand

Lagging strand Movement of the replication fork

3 5

Option 1 Option 2 if you switch the 5 and 3 ends of the template strand, then the leading strand would be on the bottom, and the lagging strand on the top.

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4. On the diagram below add in the following proteins in the correct positions. Helicase DNA Pol III core SSB PCNA (sliding clamp) Primase Ligase

Movement of the replication fork

5. Briefly summarize the palm, thumb, and finger domains of DNA polymerase, and explain how they contribute to replication. What is the role of the metal ions in the palm's activity? Palm: Contains the active site required for the synthesis reactions. Also ensures that the correct base pair is added. This occurs because of steric (shape) constraints of the active site. The metal ions in the active site serve two functions: 1) to destabilize the 3 OH so that nucleophillic attack can occur; 2) they help coordinate the phosphate groups so that the dNTP is positioned correctly for the synthesis reaction to happen. The palm also contains the proofreading exonuclease activity that removes mis-incorporated base pairs. Fingers: Also important for the synthesis reaction; they make contact with the incoming dNTP and help grip the dNTP into the correct place so the reaction can occur. The finger domain also interacts with the template strand, causing a 90 deg. bend so that only one template base is exposed in the active site at a time. This prevents any confusion as to which dNTP is supposed to be added next. Thumb: Does not participate directly in catalysis, but does interact with the newly synthesized DNA. This helps to maintain the correct positioning of the primer:template junction in the active stie, and a strong association between the polymerase and substrate. This increases the processivity of the enzyme as a whole.

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6. Describe the steps involved in the initiation of DNA replication in E. coli. What is the MAIN difference between eukaryotes and prokaryotes in the way replication is initiated? Initiator binds initiator binding sites and stimulates melting of the origin sequences. DNA helicase is then recruited and loaded by the helicase loader. As helicase unwinds the helix, primase is recruited to lay down the primer, and the rest of the replication machinery assembles at the repl. forks. In Euks, initiation requires 2 steps recognition of the initiator binding sites by ORC and formation of the pre-RC, followed by activation of the complex during S phase.

7. Eukaryotic cells and prokaryotic cells face different challenges in finishing replication. Why are the challenges different? Briefly describe the challenges for each, and how organisms solve these problems. The challenges are different because pros (most) have circular chromosomes while euks have linear. Replication of circular chromosomes results in two double-stranded circles being linked together after replication. They must be separated from one another prior to cell division this is the job of topoisomerase II. Linear chromosomes have the end-replication problem. The use of RNA primers results in a gap at the very 5 end of a linear chromosome that DNA polymerase cant fill in there is no 3 OH to add to. Euks solve this by using the enzyme telomerase a ribonucleoprotein that carries an RNA that is complementary to the DNA found at the telomeres. This RNA serves as a template to extend the 3 end of a chromosome to a point where RNA primase can add another primer and DNA polymerase can fill in the gap. Linear chromosomes that use telomerase will always have a 3 overhang because the most 5 end will never be replicated, but that isnt a problem for the repetitive telomere sequences. As long as the telomeres remain long enough that the receeding ends dont get into euchromatic portions of the chromosome. 8. The word mutation usually has a negative connotation. Why is this not true? What is the definition of mutation? Why might mutations sometimes be a good thing? Mutations are any permanent changes in the genetic material. Not every change has a discernable effect on the organisms phenotype DNA changes can be good, bad or neutral. The genome needs to be mutable because populations evolve in changing environments and need to be able to adapt thus some mutations may lead to a new and improved protein function that is beneficial to the organism.

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9. Name three types of point mutations. Using the sequence below give an example of each type write out what the sequence would look like when it harbors the particular type of point mutation you are describing. Transitions, transversions, indels 5 ATTACGGCAGTGC 3 3 TAATGCCGTCACG 3 5 ATTGCGGCAGTGC 3 3 TAACGCCGTCACG 3 5 ATTTCGGCAGTGC 3 3 TAAAGCCGTCACG 3 5 ATTACCGGCAGTGC 3 3 TAATGGCCGTCACG 3 Transition Transversion Insertion

10. Haploid yeast cells that preferentially repair double-strand breaks by homologous recombination (rather than by non-homologous end joining) are especially sensitive to agents that cause double-strand breaks in DNA. If the breaks occur in the G1 phase of the cell cycle, most cells will die; however, if the breaks occur in the G2 phase, a much higher fraction of cells survive. Why do you suppose this is? Repair by homologous recombination requires that there be two copies (homologs!) of the genetic information in the cell. Haploid cells by definition do not have two copies of each chromosome in the cell during G1. During G2, after replication has happened, the sister chromatids can function as homologs, one providing the information needed to repair the broken chromosome.

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11. For each of the following instances of DNA damage, determine the nature of the mutation that will occur if the damage is not repaired, and which DNA repair mechanism is likely to fix the problem. An example is shown. Mutational Event Example: Oxidation of the 8-carbon of guanine (oxoG) Deamination of cytosine Type of Mutation (if not repaired!) GC TA transversion CG TA transition DNA repair mechanism? Base excision repair Base excision repair

Nucleotide excision repair Pyrimidine dimer Indel or random insertion of nucs by translesion polymerase GC AT transition Direct repair by demethylase

Methylation of the oxygen of carbon 6 of guanine (O6methylguanine)