Exam 11: DNA Replication and Recombination

List four enzymes known to be involved in the replication of DNA in bacteria.

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 cluster of terms accurately reflects the nature of DNA replication in prokaryotes?

The accompanying figure diagrams DNA replication as currently believed to occur in E. coli. From specific points, arrows lead to numbers. 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?

Given the accompanying diagram, assume that a G1 chromosome (left) underwent one round of replication in ³H-thymidine and the metaphase chromosome (right) had both chromatids labeled. Which of the following replicative models (conservative, dispersive, semiconservative) could be eliminated by this observation?

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.

Compare the rate of DNA replication in prokaryotes and eukaryotes.

In general, DNA replicates semiconservatively and bidirectionally.

Which terms accurately reflect the nature of replication of the chromosome in E. coli?

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 sample A). You then wash the remaining E. coli cells and grow them for one generation in ¹⁴N, and extract the DNA from a portion of the culture (call this sample B). You let the culture grow for one more generation in ¹⁴N, and extract the DNA (call this sample C) is then subjected to ultracentrifugation. Present the centrifugation profiles that you would expect under (a) semiconservative replication and (b) conservative replication. (Note: Assume that unlabeled [¹⁴N] DNA has a density of 1.700 gm/cm³.) C). Each sample of DNA (A, B, and

Given that the nature of DNA replication in eukaryotes is not as well understood as in prokaryotes, (a) present a description of DNA (chromosome) replication as presently viewed in eukaryotes and (b) state the differences known to exist between prokaryotic and eukaryotic DNA replication.

DNA replication in vivo requires a primer with a free 3′ end. What molecular species provides this 3′ end, and how is it provided?

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

Refer to the accompanying diagram of a generalized tetranucleotide to answer questions (a) through (e). (a) Is this a DNA or an RNA molecule? (b) Place an "X" (in one of the circles in the diagram) at the 3′ end of this tetranucleotide. (c) Given that the DNA strand, which served as a template for the synthesis of this tetranucleotide, was composed of the bases 5′ - A C A G - 3′, fill in the parentheses (in the diagram) with the expected bases. (d) Suppose that one of the precursors for this tetranucleotide was a ³²P-labeled guanine nucleoside triphosphate (the innermost phosphate containing the radioactive phosphorus). Circle the radioactive phosphorus atom as it exists in the tetranucleotide. (e) Given that spleen diesterase (breaks between the phosphate and the 5′ carbon) digests the pictured tetranucleotide, which base(s) among the breakdown products would be expected to be attached to the ³²P?

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 sample A). 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 sample B). You let the culture grow for one more generation in ¹⁴N, and extract the DNA (call this sample 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, and C) 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.) C). Each sample of DNA (A, B, and

A nucleosome is a structure associated with the nuclear membrane. It helps maintain a stable relationship between the extracellular matrix and the membrane itself.

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

The discontinuous aspect of replication of DNA in vivo is caused by ________.

Telomerase is an RNA-containing enzyme that adds telomeric DNA sequences onto the ends of linear chromosomes.

Given that the origin of replication is fixed in E. coli, what signals the location of the origin?