Flashcards in BIOL #12: DNA Structure & Replication Deck (32):
Compared to all other molecules in nature, nucleic acids (DNA and RNA) are unique in their ability to direct their own replication from monomers (nucleotides)
Replication is precise, resulting in the resemblances between parents and offspring
DNA encodes a chemical language that directs the development of your biochemical, anatomical, physiological, and to some extent, behavioral traits
Since the early 1900s, biologists
- Knew chromosomes were comprised of DNA and protein
- Did not know whether genes were comprised of DNA or protein.
+ Until the 1940s, general consensus supported the hypothesis that genes were comprised of proteins because of the relative complexity and variability of proteins compared to DNA (comprised of only four different nucleotides: A, T, C, G)
DNA has directionality—one end has an exposed hydroxyl group on the 3′ carbon of deoxyribose, and the other end has an exposed phosphate group on a 5′ carbon.
- The molecule thus has a 5′ end and a 3′ end.
The role of DNA in heredity (transmission of traits from one generation to the next) was determined based on the accumulation of evidence from different experiments in the 1920s-1950s:
- Bacteria found to pick up DNA (not protein) from environment and incorporate it into their genome (transformation)
- Only DNA (not protein) found to cause viral infection in host cells
- Species found to differ in their DNA base composition (e.g. human DNA is 30.3% A while E. coli (bacteria) DNA is 26.0% A)
Watson & Crick
Once there was a consensus that DNA was the genetic material, scientists needed to determine how the structure of DNA allowed for it to be inherited (e.g. replicated)
- In the 1950s, with the aid of Rosalind Franklin’s work, Watson & Crick modeled the secondary structure of DNA
Watson and Crick proposed that two DNA strands line up in opposite directions to each other, in what is called antiparallel fashion.
- 5’ and 3’ directions of strands are in opposite directions
The antiparallel strands then twist to form a double helix.
Complementary Base Pairing
The secondary structure is stabilized by complementary base pairing:
- Adenine (A) hydrogen bonds with thymine (T).
- Guanine (G) hydrogen bonds with cytosine (C).
Necessary for both asexual (mitosis) and sexual reproduction to occur (meiosis)
DNA replication is the process by which a DNA molecule is copied
Nucleic acids (DNA and RNA) are unique biological molecules because of their ability to direct their own replication from monomers (nucleotides)
Watson & Crick originally stated the base-pairing rules (A-T, C-G) “suggested a possible copying mechanism for the genetic material”
DNA Strands Are Templates for DNA Synthesis
Watson and Crick suggested that the existing strands of DNA served as a template (pattern) for the production of new strands, with bases being added to the new strands according to complementary base pairing.
- Each strand stores the information necessary to reconstruct the other strand
Biologists then proposed three alternative hypotheses for how the old and new DNA strands interact during replication:
1) Semiconservative replication
2) Conservative replication
3) Dispersive replication
In conservative replication, the parental molecule serves as a template for the synthesis of an entirely new molecule.
In semiconservative replication, the parental DNA strands separate and each is used as a template for the synthesis of a new strand. Daughter molecules each consist of one old and one new strand.
In dispersive replication, the parent molecule is cut into sections such that the daughter molecules contain old DNA interspersed with newly synthesized DNA.
The Meselson-Stahl Experiment
In the 1950s, researchers Meselson and Stahl designed an experiment to provide more information about whether one of these hypotheses was correct.
They grew E. coli in the presence of “heavy” nitrogen (15N isotope) to label the bacteria's DNA. After many generations, they moved the bacteria to a normal 14N-containing medium and separated the DNA by density.
The densities of the resulting DNA samples supported semiconservative DNA replication as the mechanism by which the hereditable material is duplicated.
Meselson and Stahl showed that each parental DNA strand is copied in its entirety, but did not illustrate a mechanism for this process.
- The basic principle of DNA replication is conceptually simple but the actual process requires some complicated biochemical interactions
The discovery of DNA polymerase, the enzyme that catalyzes DNA synthesis, cleared the way for understanding DNA replication reactions.
DNA polymerases are the enzymes that catalyze the polymerization of DNA strands by adding nucleotides to a preexisting strand.
A critical characteristic of DNA polymerases is that they can only work in one direction. DNA polymerases can add deoxyribonucleotides to only the 3′ end of a growing DNA chain. As a result, DNA synthesis always proceeds in the 5′ → 3′ direction.
How Does Replication Begin?
A replication bubble forms in a chromosome that is actively being replicated.
- Replication bubbles grow as DNA replication proceeds, because synthesis is bidirectional.
In bacterial chromosomes, the replication process begins at a single location, the origin of replication.
Eukaryotes also have bidirectional replication but they have multiple origins of replication and thus have multiple replication bubbles.
A replication fork is the Y-shaped region where the DNA is split into two separate strands for copying.
How Is the Helix Opened and Stabilized?
Several proteins are responsible for opening and stabilizing the double helix:
- The enzyme helicase catalyzes the breaking of hydrogen bonds between the bases of the DNA strands to separate them.
- Then single-strand DNA-binding proteins (SSBPs) attach to the separated strands to prevent them from closing.
Unwinding the DNA helix creates tighter twisting and tension farther down the helix.
- The enzyme topoisomerase cuts, swivels, and rejoins the DNA downstream of the replication fork, relieving this tension.
How Is the Leading Strand Synthesized?
DNA polymerase cannot initiate synthesize of the new strand, it can only add nucleotides to the end of an already existing chain corresponding to the template strand.
- DNA polymerase requires a primer —which consists of a few RNA nucleotides bonded to the template strand—because it provides a free 3′ hydroxyl (OH) group that can combine with an incoming dNTP to form a phosphodiester bond.
- Primase is the enzyme that synthesizes a short RNA segment that serves as a primer for DNA synthesis.
The product of primase activity is called the leading strand, or continuous strand, because it leads into the replication fork and is synthesized continuously in the 5′ 3′ direction.
The 3’ end of the primer produced by primase is where RNA polymerase III attaches to start adding complementary nucleotides
The Lagging Strand
The lagging strand is part of the strand that occurs before the origin of replication. It is synthesized discontinuously in the direction away from the replication fork – its synthesis lags slightly in time because enough of the template must be exposed for synthesis to occur
The lagging strand exists because DNA synthesis must proceed in the 5' to 3' direction.
How Is the Lagging Strand Synthesized?
As with the leading strand, synthesis of the lagging strand starts when primase synthesizes a short stretch of RNA that acts as a primer. DNA polymerase III then adds bases to the 3' end of the primer.
DNA polymerase moves away from the replication fork, even though helicase continues to open the replication fork and expose single-strand DNA on the lagging strand.
The Discontinuous Replication Hypothesis
The discontinuous replication hypothesis:
- States that once primase synthesizes an RNA primer on the lagging strand, DNA polymerase might synthesize short fragments of DNA along the lagging strand, and that these fragments would later be linked together to form a continuous whole.
This hypothesis was tested by Okazaki and his colleagues.
The lagging strand is synthesized as short discontinuous fragments called Okazaki fragments.
- DNA polymerase III synthesizes a short segment of DNA then is released when it reaches the last primer
- New primer and DNA polymerase III starts again closer to the replication for
DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment and fills in the gap with DNA nucleotides.
DNA polymerase I cannot join the last nucleotides of each strand with the adjacent strand.
- The enzyme DNA ligase joins the Okazaki fragments to form a continuous DNA strand.
Because Okazaki fragments are synthesized independently and joined together later, the lagging strand is also called the discontinuous strand.
Main Differences Between Leading and Lagging Replication
The leading strand elongates continuously and only requires one primer (at the origin of replication) for DNA polymerase III to add nucleotides
The lagging strand must work away from the replication fork, thus needs to be synthesized discontinuously, in a series of segments – this process requires multiple primers and multiple DNA polymerase molecules.
Replicating the Ends of Linear Chromosomes
As the replication fork reaches the end of a linear chromosome, there is no way to replace the RNA primer from the lagging strand with DNA, because there is no available primer for DNA synthesis.
The last primer is removed, leaving a section of single-stranded DNA (lagging strand) at one end of each new chromosome.
This remaining single-stranded DNA is eventually degraded, resulting in shortening of the chromosome.
Prokaryotes have circular chromosomes so shortening of DNA does not occur
In eukaryotic organisms, the ends of the linear chromosomes have special nucleotide sequences called telomeres
- Telomeres do not contain genes but rather multiple repetition of non-coding nucleotide sequences
Telomeric DNA acts as a buffer zone to protect genes on the chromosomes from degrading
Replication of telomeres can be problematic:
- Leading-strand synthesis results in a normal copy of the DNA molecule
- Telomere on the lagging strand shortens during DNA replication.
The enzyme telomerase adds more repeating bases to the end of the lagging strand, catalyzing the synthesis of DNA from an RNA template that it carries with it.
Primase then makes an RNA primer, which DNA polymerase uses to synthesize the lagging strand. Finally, ligase connects the new sequence.
This prevents the lagging strand from getting shorter with each replication
Replication in Gametes & Somatic Cells
Telomerase is active in cells that produce gametes but somatic cells normally lack telomerase.
- The chromosomes of somatic cells have telomeres but they progressively shorten as the individual ages.
This has led biologists to hypothesize that telomere shortening has a role in limiting the amount of time cells remain in an actively dividing state
- Hypothesized that normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions somatic cells can undergo
+ Interestingly, some cancerous cells have been observed to have telomerase activity, which is why some cancerous cell lines might divide indefinitely rather than die out (called immortal cell lines).
DNA Replication Efficiency & Accuracy
Each human cell has 46 DNA molecules – one long double-helix molecule per chromosome.
- This represents ~6 billion nucleotide pairs (1000x the DNA found in bacterial cells)
- If you were to print your DNA bases in 12 pt font (A, T, G, C), the 6 billion nucleotide pairs in a diploid cell would fill 1,200 books as thick as your Campbell textbook
- It only takes a few hours for your cells to copy all of this DNA and this is achieved with very few errors!
Repairing Mistakes and Damage
DNA replication is very accurate, with an average error rate of less than one mistake per 10 billion bases.
DNA polymerase is highly selective in matching complementary bases correctly.
- As a result, DNA polymerase inserts the incorrect base only about once every 100,000 bases added.
- DNA polymerase can proofread during replication.
If mistakes remain after synthesis is complete or if DNA is damaged, repair enzymes can remove the defective bases and repair them.
How Does DNA Polymerase Proofread?
DNA polymerase can proofread its work—it checks the match between paired bases, and can correct mismatched bases when they do occur.
- DNA polymerase III can do this because one of its subunits acts as an exonuclease, an enzyme that removes deoxyribonucleotides from DNA.
If—in spite of its proofreading ability—DNA polymerase leaves a mismatched pair behind in the newly synthesized strand, a battery of enzymes are present to correct the problem.
Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete.
Mismatch repair enzymes (called nucleases) recognize the mismatched pair, remove a section of the newly synthesized strand that contains the incorrect base, and fill in the correct bases.
Repairing Damaged DNA
Similar repair mechanisms can be used if DNA is damaged. DNA can be broken or altered by various chemicals and types of radiation.
- For example, UV light can cause thymine dimers to form, causing a kink in the DNA strand.
Changes in DNA are usually corrected before they become permanent changes (mutations) perpetuated through successive replications.
The nucleotide excision repair system recognizes such types of damage. Its enzymes (nucleases) then remove the single-stranded DNA in the damaged section.
The presence of a DNA strand complementary to the damaged strand provides a template for resynthesis of the defective sequences.
Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in humans characterized by the development of skin lesions.
XP is caused by mutations of one of several nucleotide excision repair systems. These mutations mean that the cells of people with XP cannot repair DNA damaged by ultraviolet radiation.
The cells of individuals with XP are more vulnerable to UV light damage
The cells of individuals with XP have no ability to repair damaged DNA