Biology CH 14: DNA and the Gene - Synthesis and Repair
How does the nucleotide excision repair work?
1) Enzymes detect an irrgularity in DNA structure and cut the damaged strand. 2) An enzyme excises nucleotides on the damaged strand. 3) DNA polymerase fills in the gap in the 5' --> 3' direction. 4) DNA ligase links the new and old nucleoties.
replication fork
A Y-shaped region, in the corners of each replication bubble, where the parent DNA double helix is split into two single strands, which are then copied.
primer
A few nucleotides that are bonded to the template strand of DNA.
discontinous replication
A hypothesis by Reihi Okazaki and colleagues that stated that once primase synthesizes an RNA primer on the lagging strand, DNA polymerase III might synthesize short fragments of DNA along the lagging strand and that these fragments would later be linked together to form a continuous whole. The idea was that primase would add a primer to the newly exposed single-stranded DNA at intervals and that DNA polymerase III woulf then synthesize the lagging strand until it reached the fragment it produced earlier.
medium
A liquid or solid that is suitable for growing cells.
strain
A population of genetically identical individuals.
What is the problem with telomeres?
A single-stranded DNA that remains after telomeres are copied are eventually degraded, which results in the shortening of the chromosome. If this process were to continue, every chromosome would shorten by 50-100 deoxyribonucleotides on average each time DNA replication occured prior to mitosis or meiosis. Over time, linear chromosomes would be expected to disappear completely. To maintain the integrity of linear chromosomes in eukaryotes, telomere replication happens.
virulence
Ability to cause disease and death; varies; virulent strains (S) cause disease, while avirulent strains (R) do not.
semiconservative
Accepted hypothesis that DNA replication makes new DNA molecules that comprise of one old strand and on new strand.
leading strand
Also known as continuous strand, it is the product of the enzyme DNA polymerase. It leads into the replication fork and is synthesized continuously.
nucleotide excision repair
An efficient system to fix problems when genes are under constant assault (ex: from chemicals produced during aerobic metabolism, radiation, UV light that cause thymine dimer kinks, etc).
RNA polymerase
An enzyme that catalyzes the polymerization of ribonucleotides to RNA.
What happens before DNA synthesis gets under way?
An enzyme, called primase, has to synthesize a short stretch of RNA that acts as a primer for DNA polymerase. Primase is a type of RNA polymerase. Unlike DNA polymerases, primase and other RNA polymerases don't require a primer. These enzymes can simply match ribonucleotides directly by complementary base pairing on single-stranded DNA. In this way, primase creates a primer in DNA synthesis.
DNA ligase
Catalyzes the joining of Okazaki fragments in the lagging strand into a leading strand.
primase
Catalyzes the synthesis of the RNA primer in the leading strand and catalyzes the synthesis of th RNA primer on an Okazaki fragment in the lagging strand.
somatic cells
Cells that are not involved in gamete formation. Normally lack telomerase. As predicted, chromosomes of somatic cells gradually shorten w/ each mitotic division, getting progressively smaller as an individual grows and ages.
culture
Collection of cells that grows under controlled conditions-- usually suspended in a liquid medium or on the surface of a solid growth medium.
How can the enzymes involved in replicating DNA be as precise as they are?
DNA polyermase is highly selective in matching complementary bases correctly. They can also "proof read" during the synthesis process. If mistakes remain, repair enzymes can cut out the defective bases and replace them.
What happens at the start of DNA synthesis?
DNA polymerase III works only in the 5' --> 3' direction. To recognize to start synthesis, it requires both a 3' end and a single-stranded template. The single-stranded template dictates which deoxyribonucleotide should be added next, while a *primer* provides a free 3' hydroxyl group that can combine w/ an incoming dNTP to form a phosphodiester bond. Once a primer is added to a template, DNA polymerase III beings working in 5--3 direction and adds deoxyribonucleotides to complete the complementary strand in a sequence that complements the template strand. The shape of DNA polymerase III grips the DNA strand during synthesis. As DNA polymerase moves along the DNA molecule, a doughnut shaped structure behind it, called the sliding clamp, holds the enzyme in place, resulting in a leading strand.
What is the transforming factor?
DNA, not RNA or proteins.
helicase
Enzyme that catalyzes the breaking of hydrogen bonds between deoxyribonucleotides. This reaction causes thw two strands of DNA to separate.
topoisomerase
Enzyme that cuts and rejoins the DNA downstream of the replication form. They do this cutting and pasting in a way that undoes twists and knots. They relieve the twisting stress that is induced by helicase / opening of the helix.
telomerase
Enzyme that is involved in replicating telomeres. It catalyzes the synthesis of DNA from an RNA template. The enzyme carries and RNA molecule with it that acts as built-in template. Telomerase catalyzes the addition of deoxyribonucleotides to the ends of chromosomes. The deoxyribonucleotides that are added are complementary to the ribonucleotides in telomerase's built-in RNA template. Adds additional repeats. Now, primase, DNA polymerase, and ligase can synthesize the lagging strand in the 5--3 direction, restoring the original length of the chromosomes and prevent it from getting shorter.
DNA polymerase
Enzyme that polymerizes deoxyribonucleotides to DNA. This protein catalyzes DNA synthesis. Organisms contain several types of DNA polymerases.
DNA polymerase III
Extends the leading strand and extends an Okazaki fragment in the lagging strand.
sliding clamp
Holds DNA polymerase in place during strand extension in both the leading and lagging strand.
How do bacteria and eukaryotes differ in their origin of replication?
In most bacteria, chromosomes are circular and there's a single point of origin (replication bubble) during DNA replication. Most eukaryotes have linear chromosomes that contain several to many points of DNA synthesis.
deoxyribonucleoside triphosphate (dNTP)
Monomers that are added to the growing strand of DNA. The N in dNTP stands for any of the four DNA bases: adenine, thymine, guanine, or cytosine. Because they have three phosphate groups close together, dNTPs have high potential energy-- high enough to make the formation of phosphodiester bonds in a growing DNA strand exergonic, when polymerization rxns are endergonic.
Mismatch repair
Occurs when mismatched bases are corrected after DNA synthesis is complete. If DNA polymerase leaves a mismatched pair behind in the DNA sequence by mistake, a battery of enzymes springs into action to correct the problem. The proteins recognize the mismatched pair, remove a section of the newly synthesized strand that contains the incorrect base, and fill in the correct bases.
What are the characteristics of the leading strand?
Primase, DNA polyermase III, and sliding clamp.
What are the characteristics of the lagging strand?
Primase, DNA polymerase III, sliding clamp, DNA polymeras I, and DNA ligase.
proofread
Process where if the wrong base is added during DNA synthesis, DNA polymerase III removes the mismatched base that was just added, and then proceeds with synthesis. This ability is the same for prokaryotic and eukaryotic DNA polymerases. Proofreading reduces a DNA polymerase's error rate to about 1 mistake per 10 million bases. Accurate enough? No. Mismatch repair is needed.
single-strand DNA-binding proteins (SSBPs)
Proteins that attach to separated DNA strands and prevent them from snapping back into a double helix. Function: stablize single-stranded DNA.
xeroderma pigmentosum (XP)
Rare autosomal recessive disease in humans. Individuals w/ XP are extremely sensitive to UV light. Nucleotide exicison repair is virtually nonexistant in XP individuals.
DNA polymerase I
Removes the RNA primer in the lagging strand and replaces it with DNA.
Okazaki fragments
Short pieces of DNA on the lagging strand. In eukaryotes, they are 100-200 base pairs long, though a chromosome typically contains millions of base pairs.
origin of replication
Specific sequence of bases where replication bubbles form in chromosomes.
lagging strand
Strand that is synthesized in the opposite direction of the replication fork.
What is an important thing to recognize about telomerase?
Telomerase isn't active in most types of cells. In humans, for example, active telomerase is found primarily in the cells of repoductive organs-- specfically, in the cells that eventually undergo meiosis and produce gametes. As a result, gametes contain chromosomes of normal length.
Hershey-Chase experiment
Test to find out if viral genes consist of DNA or protein. Involves interacting viruses that have radioactive sulfur (found in proteins) or radioactive phosphorus (found in DNA) with a cuture of E. coli. Agitate culturs in blender to separate empy viral protein coats ("ghosts" from the bacterial cells in each culture. Radioactive DNA was found in the pellet cel,l while radioactive protein was found in the solution that indicates the ghost. DNA is the hereditary material.
Why is DNA polymerases selective?
The correct base pairings in DNA (A-T and G-C) are energetically the move favorable of all possibilities for the pairing of ntriogen-containing bases. As DNA polyermase marches along a parent DNA template, hydrogen bonding occurs between income deoxyribonucleotides and the ones already on the template strand. This enzyme inserts the incorrect deoxyribonucleotide only ~1 every 100,000 bases added.
genome
The entire complement of DNA in cells.
How do eukaryotes and bacteria & archaea compare in DNA synthesis?
The leading and lagging strands of DNa molecules are copied efficiently by the same enzymes. Difference: replicating the ends of chromosomes. Bacteria & archaea do not have the end-replication problem that eukaryotes have in telomeres.
telomere
The region at the end of a linear chromosome.
complementary base pairing
The specific pairing rules for hydrogen bonding of nitrogen containing bases.
What is the critical point about DNA polymerases?
They can work in only 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.
What do helicase and SSBPs combine to do?
They open up the double helix and make both strands of DNA available for copying.
