Exam 11: DNA Replication and Recombination

In general,DNA replicates semiconservatively and bidirectionally.

Describe the function of the RecA protein.

In ligase-deficient strains of E.coli,DNA and chromosomal replication are unaltered because ligase is not involved in DNA replication.

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.

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,and extract the DNA from a portion of the culture (call this sampleB).You let the culture grow for one more generation in ¹⁴N,and extract the DNA (call this sampleC)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.)Each sample of DNA (A,B,andC).

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

DNA polymerase III adds nucleotides ________.

DNA polymerase I is thought to add nucleotides ________.

What primary ingredients,coupled with DNA polymerase I,are needed for the in vitro synthesis of DNA?

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

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.

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?

Compare the rate of DNA replication in prokaryotes and eukaryotes.

Assume that you were growing cells in culture and had determined the cell-cycle time to be 24 hours.You introduce ³H thymidine and prepare autoradiographs of metaphase chromosomes after 48 hours.Of the chromosomes that are labeled,you expect two classes: one class that had completed one S phase in the label,and a second class that had completed a cellular division and an additional S phase in the label.Draw the DNA (double-stranded)labeling pattern for each chromosome that you would expect to find in these two types of metaphase chromosomes.(Use a broken line {- - -} for labeled single strands of DNA and a solid line for unlabeled single strands of DNA.) (a)metaphase chromosome having replicated once in label (b)metaphase chromosome having gone through two S phases in label

The Meselson and Stahl experiment provided conclusive evidence for the semiconservative replication of DNA in E.coli.What pattern of bands would occur in a CsCl gradient for conservative replication?

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?

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

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 occurs in the 5′ to 3′ direction; that is,new nucleoside triphosphates are added to the 3′ end.

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