Exam 10: DNA Replication and Recombination

In general,DNA replicates semiconservatively and bidirectionally.

Assume that you grew a culture of E.coli for many generations in medium containing ¹⁵N (from the ammonium ion),a heavy isotope of nitrogen.You extract DNA from a portion of the culture and determine its density to be 1.723 gm/cm³ (call this sampleA).You then wash the remaining E.coli cells and grow them for one generation in ¹⁴N,extract the DNA from a portion of the culture,and determine its density to be 1.715 gm/cm³ (call this sampleB).You let the culture grow for one more generation in ¹⁴N and extract the DNA (call this sampleC)Each sample of DNA (A,B,and C)is then heated to completely denature the double-stranded structures,cooled quickly (to keep the strands separate),and subjected to ultracentrifugation.Present the centrifugation profiles for heat-denatured DNA (samples A,B,andC)that you would expect.Use the graph below.(Note: Although not the case,assume that single-stranded DNA has the same density as double-stranded DNA.)

Below is a diagram of DNA replication as currently believed to occur in E.coli.Arrows start from numbers and end at specific points.Answer the questions relating to the locations specified by the numbers. (1)Which end (5' or 3')of the molecule is here? (2)Which enzyme is probably functioning here to deal with supercoils in the DNA? (3)Which enzyme is probably functioning here to unwind the DNA? (4)Which nucleic acid is probably depicted here? (5)What are these short DNA fragments usually called? (6)Which enzyme probably functions here to couple these two newly synthesized fragments of DNA? (7)Is this strand the leading or lagging strand? (8)Which end (5' or 3')of the molecule is here?

Compare the rate of DNA replication in prokaryotes and eukaryotes.

During DNA replication,what is the function of RNA primase?

An endonuclease is involved in removing bases sequentially from either end of DNA.

G-quartets are G-rich single-stranded tails that loop back on themselves forming G-G double-stranded sections.Such looping is involved in aligning chromosomes for homologous recombination.

What is the name of the replication unit in prokaryotes,and how does it differ in eukaryotes?

As unwinding of the helix occurs during DNA replication,tension is created ahead of the replication fork.Describe the nature of this tension and state the manner in which it is resolved.

DNA replicates conservatively,which means that one of the two daughter double helices is "old" and the other is "new."

Briefly describe what is meant by the term autoradiography and identify a classic experiment that used autoradiography to determine the replicative nature of DNA in eukaryotes.

DNA polymerase III adds nucleotides ________.

Assume that a culture of E.coli was grown for approximately 50 generations in ¹⁵N (provided in the medium in the ammonium ion),which is a heavy isotope of nitrogen (¹⁴N).You extract the DNA from the culture,and it has a density of 1.723 gm/cm³ (water = 1.00 gm/cm³).From the literature,you determine that DNA containing only the common form of nitrogen,¹⁴N,has a density of 1.700 gm/cm³.Bacteria from the ¹⁵N culture were washed in buffer and transferred to ¹⁴N medium for one generation immediately after which the DNA was extracted and its density determined. (a)What would be the expected density of the extracted DNA? (b)After you heat the extracted DNA until it completely denatures (95°C for 15 minutes),what would you expect the density of the DNA in the denatured extract to be? For the purposes of this question,assume that DNA has the same density regardless of whether it is single- or double-stranded. (c)Assuming that the molar percentage of adenine in the extracted DNA was 20%,what would be the expected molar percentages of the other nitrogenous bases in this DNA? (d)Assume that a fraction of the extracted DNA was digested to completion with the enzyme snake venom diesterase.This enzyme cleaves between the phosphate and the 3' carbon.Present a "simplified" diagram that would illustrate the structure of the predominant resulting molecule.

Which structural circumstance in DNA sets up the requirement for the semidiscontinuous nature of its replication?

Chromatin assembly factors (CAFs)move along with the replication fork and assemble new nucleosomes.

Meselson and Stahl determined that DNA replication in E.coli is semiconservative.What additive did they initially supply to the medium in order to distinguish "new" from "old" DNA?

What term is used to describe genetic exchange at equivalent positions along two chromosomes with substantial DNA sequence homology?

Describe the function of the RecA protein.

Assume that you are microscopically examining mitotic metaphase cells of an organism with a 2n chromosome number of 4 (one pair metacentric and one pair telocentric).Assume also that the cell passed through one S phase labeling (innermost phosphate of dTTP radioactive)just prior to the period of observation.Assuming that the circle below represents a cell,draw its chromosomes and the autoradiographic pattern you would expect to see.

Each of the following terms refers to the replication of chromosomes.Describe the role (relationship)of each in (to)chromosome replication. (a)Okazaki fragment (b)lagging strand (c)bidirectional