FALL 2012 PCB 3063 Objectives for Exam 3 DNA Replication Chapter 10 Complementary base pairing underlies DNA replication In this section the historical evidence that DNA replication is semiconversative is presented and the enzymology of DNA replication is described Some important differences between DNA replication between prokaryotes and eukaryotes are introduced Q Be able to describe and interpret the results of the Meselson Stahl experiment which showed that DNA replication is semiconservative Meselson Stahl took E coli labeled with 15N and added it to 14N medium o over several generations the density bands showed that replication and allowed it to replicate must have consisted of one old strand and one new strand For more details see pg 206 in the text Be able to diagram the results of their experiment and what they would have obtained had replication proved to be conservative or dispersive Q Know the requirements of DNA polymerase template primer direction of synthesis what nucleotides are used and the reaction by which it makes DNA Formation of a phosphodiester bond involving the phosphate group on one nucleotide and the 3 OH on ribose deoxyribose on the other nucleotide adds dNTPs to the 3 end of DNA 5 to 3 synthesis equires a DNA template requires a primer can be DNA or RNA You should know this already Q Know what the 5 and 3 ends of a nucleic acid refer to Q Know the following terms Replication origin the sequence in the genome at which replication is initiated Bidirectional replication replication proceeds at the same time in two opposite directions at each replication fork Replication bubble DNA strands separate and form a bubble Replication fork structure has two branching prongs each one made up of a single strand of DNA These two strands serve as the template for the leading and lagging strands which will be created as DNA polymerase matches complementary nucleotides to the templates Leading strand The leading strand template is the strand of DNA being replicated continuously It is the strand that is being continuously polymerized toward the replication fork All DNA synthesis occurs 5 3 The original DNA strand must be read 3 5 to produce a 5 3 nascent strand Lagging strand The lagging strand grows in the direction opposite to the movement of the growing fork It grows away from the replication fork and it is synthesized discontinuously Because the strand is growing away from the replication fork it must be replicated in fragments because the Primase that adds the RNA primer has to wait until the fork opens to be able to add the primer The RNA Polymerase reaches the origin of replication and stops replication until a new RNA primer is placed Okazaki fragment are short molecules of single stranded DNA that are formed on the lagging strand during DNA replication Q Know the two main DNA polymerase enzymes of E coli I III how they are similar and how they are different in terms of their enzymatic activities and know what job each one does in the cell leading and lagging strand synthesis versus joining the Okazaki fragments proof reading removal of primers DNA repair Pol III synthesizes the leading and the lagging strand has 3 5 exonuclease Pol I completes the lagging strand removes primers and fills in any gaps DNA repair has 3 5 exonuclease and 5 3 Q Know what the exonuclease activities are of DNA polymerase I and III and what the jobs of these exonuclease activities are 3 5 exonuclease proof reading function 5 3 exonuclease primer removal Q Be able to draw the replication of DNA at a single replication fork showing the location and of helicase primase DNA polymerases and the direction of synthesis of leading and lagging strands Be able to correctly indicate the position of the 5 and 3 ends of each of the DNA molecules undergoing replication Leading strand is continuous lagging strand is in short fragments Okazaki fragments Replication proceeds in two opposite directions at the same time at each replication fork Q Be able to explain how synthesis of the lagging strand is completed which enzymes are involved Know that the primers for DNA replication are short pieces of RNA Completion of replication requires the removal of the RNA primers and joining of the Okazaki fragments DNA Pol I removes the primers DNA ligase joins the fragments because it is capable of catalyzing the formation of the phosphodiester bond that seals the discontinuously synthesized bonds Q Understand the problem of chromosome shortening that results when linear DNA as in eukaryotic chromosomes is replicated and be able to explain how eukaryotes solve this problem know what telomeres are and what the enzyme telomerase does and what the unique feature of telomerase are reverse transcriptase carries its own RNA template Telomeres ends of eukaryotic chromosomes Telomere onto the 3 end o They are maintained by the enzyme telomerase o Telomerase extends the ends of the chromosomes its adds the o Telomerase has its own RNA which is complementary to the o Telomerase is a reverse transcriptase enzyme that copies RNA into telomere repeat and serves as the template single stranded DNA Chromatin and DNA Sequence Organization Chapter 11 Q Be able to define chromatin and describe its structure nucleosomes histone proteins plus associated DNA Chromatin complex of DNA RNA histones and nonhistone proteins that make up uncoiled chromosomes characteristic of the eukaryotic interphase nucleus o As the cell cycle progresses most cells reenter mitosis where the chromatin coils into visible chromosomes once again less positively charged histones The associated proteins are divided in basic positively charged histones and o Histones contain large amounts of the positively charged AA s lysine and arginine making it possible for them to bind to the phosphate groups of the nucleotides which are negatively charged Nucleosome beads on a string a nuclear complex consisting of 4 pairs of histone molecules an octamer of H2a H2b H3 and H4 wrapped by 2 turns of a DNA molecule The major structure associated with the organization of chromatin in the nucleus o Each repeating nucleosome is associated with about 200bp DNA Q Know the difference between euchromatin and heterochromatin in terms of its structure composition cytosine methylation of DNA histone tail acetylation and transcriptional activity Euchromatin uncoiled true chromatin or chromosomal regions that are relatively uncoiled during the interphase portion of the cell cycle o
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