Molecular Biology Wk 8 Flashcards
DNA Structure and Functions
Except in some viruses, DNA serves as the genetic material in all living organisms on Earth.
■According to the Watson–Crick model, DNA exists in the form of a right-handed double helix.
■The strands of the double helix are antiparallel and are held together by hydrogen bonding between complementary nitrogenous bases.
■The structure of DNA provides the means of storing and expressing genetic information.
Evidence Favoring DNA as the Genetic Material Was First Obtained during the Study of Bacteria and Bacteriophages.
Fred Griffith was studying Streptococcus pneumoniae (pneumococcus), a bacterium that causes pneumonia. As antibiotics had not yet been discovered, infection with this organism was usually fatal. Griffith’s critical experiment (Figure) involved an injection into mice of living IIR (avirulent) cells combined with heat-killed IIIS (virulent) cells. Griffith showed that heat-killed, infectious bacteria can transform harmless, living bacteria into pathogenic ones. Later researchers prepared an extract from the disease-causing S strain of pneumococci and showed that the “transforming principle” that would permanently change the harmless R-strain pneumococci into the pathogenic S strain is DNA. This was the first evidence that DNA could serve as the genetic material.
DNA replication
Genetic continuity between parental and progeny cells is maintained by
semiconservative replication of DNA,as predicted by the Watson–Crick model.
■ Semiconservative replication uses each strand of the parent double helix as a template, and each newly replicated double helix includes one “old” and one “new” strand of DNA.
The complementarity of DNA strands allows each strand to serve as a template for synthesis of the other
The Meselson–Stahl experiment
In 1958, Matthew Meselson and Franklin Stahl published the results of an experiment providing strong evidence that semiconservative replication is the mode used by bacterial cells to produce new DNA molecules.
Meselson–Stahl experiment
After one generation, the isolated DNA was present in only a single band of intermediate density —the expected result for semiconservative replication in which each replicated molecule was composed of one new 14N-strand and one old 15N-strand (Figure). This result was not consistent with the prediction of conservative replication, in which two distinct bands would occur; thus this mode may be rejected. After two cell divisions, DNA samples showed two density bands—one intermediate band and one lighter band corresponding to the 14N position in the gradient.
Similar results occurred after a third generation, except that the proportion of the lighter band increased. This was again consistent with the interpretation that replication is semiconservative.
Semiconservative Replication in Eukaryotes
The Taylor–Woods–Hughes experiment, demonstrating the semiconservative mode of replication of DNA in root tips of Vicia faba.
(a) An unlabeled chromosome proceeds through the cell cycle in the presence of 3H-thymidine. As it enters mitosis, both sister chromatids of the chromosome are labeled, as shown, by autoradiography. After a second round of replication (b), this time in the absence of 3H- thymidine, only one chromatid of each chromosome is expected to be surrounded by grains. Except where a reciprocal exchange has occurred between sister chromatids (c), the expectation was upheld.
The micrographs are of the actual autoradiograms
obtained in the experiment.
Origins, Forks, and Units of Replication
DNA replication begins at the origin of replication.
A replicon is the length of DNA that is replicated following one initiation event at a single origin.
Replication Forks
Two Replication Forks Form at Each Replication Origin
DNA molecules in the process of being replicated contain Y- shaped junctions called replication forks. Two replication forks are formed at each replication origin (Figure 6–8). At each fork, a replication machine moves along the DNA, opening up the two strands of the double helix and using each strand as a template to make a new daughter strand. The two forks move away from the origin in opposite directions, unzipping the DNA double helix and replicating the DNA as they go (Figure). DNA replication in bacterial and eukaryotic chromosomes is therefore termed bidirectional. The forks move very rapidly—at about 1000 nucleotide pairs per second in bacteria and 100 nucleotide pairs per second in humans. The slower rate of fork movement in humans (indeed, in all eukaryotes) may be due to the difficulties in replicating DNA through the more complex chromatin structure of eukaryotic chromosomes.
7 key issues that must be resolved during DNA replication:
• Unwinding of the helix
• Reducing increased coiling generated during unwinding
• Synthesis of a primer for initiation
• Discontinuous synthesis of the second strand
• Removal of the RNA primers
• Joining of the gap-filling DNA to the adjacent strand
• Proofreading
Continuous and Discontinuous DNA Synthesis
The two strands of a double helix are antiparallel to each other—that is, one runs in the 5’ to 3’ direction, while the other has the opposite 3’ to 5’ polarity. Because DNA Pol III synthesizes DNA in only the 5’ to 3’ direction, synthesis along an advancing replication fork occurs in one direction on one strand and in the opposite direction on the other. As a result, as the strands unwind and the replication fork progresses down the helix (Figure), only one strand can serve as a template for continuous DNA synthesis. This newly synthesized DNA is called the leading strand. As the fork progresses, many points of initiation are necessary on the opposite DNA template, resulting in discontinuous DNA synthesis of the lagging strand.
Opposite polarity of synthesis along the two strands of DNA is necessary because they run antiparallel to one another, and because DNA polymerase III synthesizes in only one direction (5’ to 3’). On the lagging strand, synthesis must be discontinuous, resulting in the production of Okazaki fragments. On the leading strand, synthesis is continuous.
RNA primers are used to initiate synthesis on both strands.
A Coherent Model Summarizes DNA Replication
At the advancing fork, a helicase is unwinding the double helix. Once unwound, single-stranded binding proteins associate with the strands, preventing the reformation of the helix.
In advance of the replication fork, DNA gyrase functions to diminish the tension created as the helix supercoils. Each of the core enzymes of DNA Pol III holoenzyme is bound to one of the template strands by a sliding DNA clamp.
Continuous synthesis occurs on the leading strand, while the lagging strand must loop around in order for simultaneous (concurrent) synthesis to occur on both strands. Not shown in the figure,
but essential to replication on the lagging strand, is the action of DNA polymerase I and DNA ligase, which together replace the RNA primers with DNA and join the Okazaki fragments, respectively.
Polymerase Direction
To elongate a polynucleotide chain, DNA polymerase III requires a primer with a free 3’-OH group.
Eukaryotic DNA Synthesis
In eukaryotic cells:
➢There is more DNA than prokaryotic cells
➢The chromosomes are linear
➢The DNA is complexed with proteins
➢Eukaryotic chromosomes contain multiple origins of replication to allow the genome to be replicated in a few hours.
Table: Differences between prokaryotic and eukaryotic replication
LOOK AT GOODNOTES TABLE PPT 8
Initiation at Multiple Replication Origins
To facilitate the rapid synthesis of large quantities of DNA, eukaryotic chromosomes contain multiple replication origins. Eukaryotic replication origins not only act as sites of replication initiation, but also control the timing of DNA replication.
These regulatory functions are carried out by a complex of more than 20 proteins, called the prereplication complex (pre-RC), which assembles at replication origins. In the early G1 phase of the cell cycle, replication origins are recognized by a six-protein complex known as an origin recognition complex (ORC), which tags the origin as a site of initiation of replication.
