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BIO 202

I.

Chapter 15: DNA Structure, Replication, and Repair

Proof that DNA is the hereditary material A. Hershey and Chase’s T2 virus experiments 1. T2 attaches to host cell, leaves a capsule outside, and injects a material inside.

2.

The injected material from the virus must be the hereditary material - analysis showed that the capsule is protein and the injected material is DNA II. Mendelson and Stahl revealed that DNA replication is semi-conservative A.

Three different models of DNA replication were proposed initially. 1. Semiconservative replication model - two original strands separate, each serving as a template for a new strand. 2. Conservative replication model - the parent DNA molecule is unchanged; the daughter molecule would consist of two entirely new DNA strands. 3. Dispersive replication model - each DNA strand in a new daughter DNA molecule would consist of a mixture of parent and daughter DNA. B. Meselson and Stahl experiment proved the semiconservative model. 1. Distinguished parent DNA from new DNA by tagging it with 15N. 2. Experimental strategy 3. Results supported the semiconservative model of DNA replication. III. A Model for DNA Synthesis A. Observations of How DNA Synthesis Occurs in Vivo 1. DNA polymerases add dNTPs only to the 3’ end of an existing strand. 2. DNA synthesis always proceeds in the 5’ to 3’ direction. 3. DNA polymerase can add only to an existing 3’-OH group; therefore, it needs a short oligonucleotide primer to begin replication. 4. Electron micrographs show DNA replication is bidirectional. a. Replication is initiated at an origin of replication, forming a “bubble”. b. Replication proceeds in both directions at the same time. c. Each side of the bubble forms a Y-shaped replication fork. B. Opening the Helix – helicases and topoisomerases at the replication fork 1. Helicases open the replication fork by breaking hydrogen bonds between DNA strands; single strands are stabilized by single-stranded binding proteins. 2. Topoisomerases relieve tension in the DNA molecule. C. Synthesis of the Leading Strand 1. Primase builds a short RNA primer that is complementary to the parent strand. 2. DNA polymerase III adds dNTPs to the 3’ end of the primer. 3. This process occurs continuously (without interruption) on the leading strand. D. Synthesis of the Lagging Strand 1. The other parent strand is antiparallel to the leading strand, so after primase adds a primer, DNA pol III must work away from the replication fork on this strand, called the lagging strand. 2. When helicase expands the replication fork, a new segment of parent strand is exposed. a. A new primer must be made to initiate replication in this section. b. This results in discontinuous replication on the lagging strand. 3. Okazaki tested this hypothesis in a pulse-chase experiment: a. Added radioactive thymidine in a brief pulse to E. coli cells in culture. b. Followed the pulse with a large “chase” of nonradioactive thymidine. c. Isolated the DNA from the cells and centrifuged it.

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BIO 202

Chapter 15: DNA Structure, Replication, and Repair

d.

4.

Result: detected some radioactive thymidine in short (~1 kb) fragments of DNA. e. Conclusion: The lagging strand is synthesized in short, discontinuous fragments (“Okazaki fragments”). Removing the primers and joining the fragments a. DNA polymerase I removes the primers at the start of each fragment.

b. IV.

DNA ligase catalyzes the formation of a phosphodiester bond between the 3’-OH of one fragment, and the 5’-P of the next fragment.

Replicating “Telomeres”, the Ends of Linear Chromosomes A.

V.

A problem arises during replication of the telomeres. 1. Replication of the leading strand can proceed to the end of the chromosome. 2. Replication of the lagging strand cannot proceed to the end of the chromosome. a. Primase can add a primer near the end of the chromosome, but once the primer is removed, DNA polymerase cannot replace the primer because there is no 3’—OH group onto which it can add. b. As a result, there is a bit of single-stranded DNA, a 3’ overhang, on each end of the chromosome, that is not replicated and instead gets degraded. d. Over time and after many replications, this causes the ends of chromosomes to get chewed away. B. Some cells express telomerase, an enzymes that can replicate telomeres 1. Telomerase carries an RNA template that lines up at the end of a chromosome. 2. This allows the normal replication machinery to come in and replicate the lengthened telomere. 3. In humans, telomerase is active in germ-line cells, but not in most somatic cells. (1) Telomere shortening may eventually cause somatic cells to stop dividing altogether and contribute to aging. (2) Telomerase activation in somatic cells may induce uncontrolled cell division and even cancer. Correcting Mistakes in DNA A. Some mistakes come from errors in DNA replication 1. DNA polymerase inserts incorrect nucleotides about once every 1000 bases. 2. Some mismatches are corrected immediately by DNA polymerases’ proofreading ability. 3. Other mismatches are repaired by specific mismatch repair systems (example: methylation-directed mismatch repair in bacterial cells) B. Other Mistakes Occur When DNA is damaged 1. The bonds that hold DNA together are susceptible to spontaneous or induced breakage. X-rays, gamma rays, and UV rays in particular can damage DNA and lead to replication mistakes that change the DNA sequence. 2. Enzymes that repair damaged DNA are called excision repair systems. a. These enzymes recognize sites of damage by identifying misshapen segments of the double helix. b. These enzymes then excise (cut out) the stretch of nucleotides surrounding the site of damage. c. The undamaged, complementary strand serves as a template for the replacement of the damaged sequence. Page 2

BIO 202

C.

Chapter 15: DNA Structure, Replication, and Repair

Defects in the Systems That Correct Errors in DNA Can Cause Disease 1. Example: mismatch repair defects and Herditary Nonpolyposis Colorectal Cancer (HNPCC) 2. Example: excision repair defects and xeroderma pigmentosum (XP).

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