L11 - DNA structure and replication

Question 1

What are the four fundamental requirements for genetic material?

Answer 1

1. Contain complex information, 2. Replicate faithfully, 3. Encode the phenotype, 4. Have the capacity to vary

Question 2

What does it mean that genetic material must contain complex information?

Answer 2

Instructions for the traits and functions of an organism.

Question 3

What does it mean that genetic material must replicate faithfully?

Answer 3

Every organism begins as a single cell. To produce a complex multicellular organism like yourself, the single cell must undergo billions of cell divisions. At each division, the genetic instructions must be accurately transmitted to descendent cells. And when organism reproduce, and pass genes to their progeny, the genetic material must be copied with fidelity.

Question 4

What does it mean that genetic material must encode the phenotype?

Answer 4

The genetic material must be able to encode for traits. There must be mechanisms for genetic instructions to be transformed into a phenotype.

Question 5

What does it mean that genetic material must have the capacity to vary?

Answer 5

Different species and different individuals within a species differ in their genetic makeup and phenotypes.

Question 6

What is the DNA structure of nucleotides?

Answer 6

Consists of ionised hydroxyl groups, phosphate group, deoxyribose sugar. The subunits of DNA are its nucleotides. A nucleotide consists of three components : a five-carbon sugar, a base, and one or more phosphate groups. In DNA, the sugar is deoxyribose. The phosphate group has two negative charges on its oxygen atoms; this is due to the ionisation of the hydroxyl groups attached to the phosphorus atom at cellular pH. This negative charge is important in DNA analysis (e.g. gel electrophoresis)

Question 7

What is the DNA structure of bases and strands?

Answer 7

A nucleotide normally contains one of four kinds of bases, denoted as A, T, C, G. Two of these bases are double ring structures known as purines - adenine and guanine. Two other are single-ring structures known as pyrimidines - thymine and cytosine. In a nucleic acid, each nucleotide sugar is linked to the phosphate group of the neighbouring nucleotide. The C-O-P-O-C linkage connecting two nucleotides is known as a phosphodiester bond. The linkage is stable and can withstand changes in pH and temperature that might break weaker bonds. A strand of DNA has polarity, meaning that one end differs from the other. At the top of the figure, you can see the free 5’ phosphate group; at the bottom, notice the free 3’ hydroxyl group. By convention, if a DNA sequence is not specified as 5’ or 3’, the end at the left (or top) is the 5’ end. Therefore the sequence in this figure is ACGT.

Question 8

What is the DNA structure of base pairing?

Answer 8

Two antiparallel strands. Base pairings of A-T and C-G. The bases A-T and C-G are said to be complementary. Base stacking attraction increases stability. Hydrogen bonds between bases hold the two strands together. Hydrogen bonds are relatively weak bonds - the two strands can be separated relatively easily

Question 9

What is the DNA structure double helix?

Answer 9

Two strands of DNA coil into a double helix, bases can come in any order, allowing the DNA to contain information and to have the capacity to vary. Structure was discovered by Watson, Crick and others including Franklyn in 1950s. The number of possible base sequences in a DNA molecule of 133 nucleotides in length is equal to the estimated number of electrons, protons and neutrons in the entire universe.

Question 10

What is the RNA structure?

Answer 10

Ribose has hydroxyl (-OH) group where deoxyribose has (-H). Uracil (pyrimidine base pair which replaces Thymine) has a hydrogen (-H) where thymine has a methyl (-CH3) group

Question 11

DNA vs. RNA structure?

Answer 11

Sugar - deoxy vs ribose
Bases - ATCG vs AUCG
5’ end - Monophosphate vs triphosphate
Size - very large vs smaller
Strands - double vs single

Question 12

How does DNA pack?

Answer 12

DNA forms supercoils which allow it to take up less space. Positive supercoiling - twisted in the same direction as the DNA double helix (overwound). Negative supercoiling - twisted in the opposite direction of the DNA (underwound). Most cellular DNA is negatively supercoiled, which eases separation of the strands for replication and transcription and allows packing of DNA into a smaller space than relaxed DNA can fit into.

Question 13

What is DNA packing in Prokaryotes?

Answer 13

The DNA double helix in the circular bacterial chromosome is folded by DNA-binding proteins into supercoil loops that protrude from a denser core (the chromosome is packed like a bottle brush)

Question 14

What is DNA packing in Eukaryotes?

Answer 14

Lots of DNA to pack up, the DNA is wrapped around histones to form nucleosomes. Nucleosomes are condensed into chromatin which is packed into chromosomes.

Question 15

What is DNA packing in Nucleosomes?

Answer 15

Approx 150 nucleotides wrapped twice around 8 histone proteins (2 each of histones H2A, H2B, H3, H4). Histones proteins are rich in positively charged amino acids, which enable them to form ionic bonds with the negatively charged sugar-phosphate backbone of the DNA. This removes the negative charge of the DNA so helps with packing. The structure needs to be disassembled for DNA replication/transcription.

Question 16

What is a nucleosome?

Answer 16

Flattened sphere consisting of approximately 150 nucleotides wrapped twice around two molecules each of histones H2A, H2B, H3, H4.

Question 17

What are histone proteins rich in?

Answer 17

Positively charged amino acids lysine and arginine, which enable them to form ionic bonds with negatively charged sugar-phosphate backbone of the DNA.

Question 18

What is DNA and RNA secondary structure?

Answer 18

Variation in secondary structures can occur depending on the conditions they are under and on the base sequences. Can influence DNA mutation rates. Triple-coiled H-DNA breaks more readily than double-stranded DNA, leading to higher mutation rates. DNA sequences capable of adopting H-DNA conformation are common in mammals, and evidence suggests that H-DNA occurs under natural conditions. Quadruplex structures involving four strands of DNA can also occur under some conditions. Some secondary structures can make DNA sequencing challenging.

Question 19

What happens is Semiconservative replication?

Answer 19

Occurs whereby the two DNA strands separate and are each used as a template for a new strand. Parental strands serve as the templates for the daughter strands

Question 20

What happens in DNA polymerisation?

Answer 20

DNA polymerisation occurs only in the 5’-3’ direction and is catalysed by DNA polymerase. DNA polymerase is highly conserved among species. Its basic function is to synthesise a new DNA strand from an existing template. Most DNA polymerases also correct mistakes in replication.

Question 21

What happens in DNA replication Step 1?

Answer 21

The two daughter strands are made differently as they are antiparallel. SInce they can be elongated only at the 3’ end.

Question 22

What happens in DNA replication Step 2?

Answer 22

The leading strand has its 3’ end pointing toward the replication fork; it is synthesised as one long, continuous polymer as the parental strand is unwound.

Question 23

What happens in DNA replication Step 3?

Answer 23

The lagging strand has its 3’ end pointed away from the replication fork; it is synthesised in short, discontinuous pieces.

Question 24

What happens in DNA replication Step 4?

Answer 24

As parental strand is unwound, a new piece is initiated at intervals and each new piece is elongated until it reaches the piece in front of it.

Question 25

What happens in DNA replication Step 5?

Answer 25

These short pieces are referred to as Okazaki fragments

Question 26

What are the four enzymes involved in DNA replication?

Answer 26

RNA primase, DNA polymerase, a different DNA complex, DNA ligase.

Question 27

What does RNA primase do?

Answer 27

Synthesises a short piece of RNA complementary to the DNA template. DNA polymerase cannot begin a new strand on its own so, each new DNA strand must begin with a short RNA primer.

Question 28

What does DNA polymerase do?

Answer 28

Elongates the primer, adding successive DNA nucleotides to the 3' end of the growing strand.

Question 29

What does a different DNA polymerase complex do?

Answer 29

Removes the RNA primer and replaces it with DNA nucleotides.

Question 30

What does DNA ligase do?

Answer 30

Joins the okazaki fragments together

Question 31

What occurs in proofreading in DNA replication?

Answer 31

DNA polymerase can detect the number of hydrogen bonds forming between the base on the template strand and the new base. If an error is detected, DNA polymerase removes it the base and inserts the correct one. Most DNA polymerases can correct their own error through proofreading.

Question 32

What happens in DNA replication in bacteria with circular DNA?

Answer 32

Single origin of DNA replication, but two replication forks. Circular chromosome, origin of replication, replication starts at the origin and moves around the circular chromosome in both directions.

Question 33

What happens in DNA replication in Eukaryotes?

Answer 33

In eukaryotes, DNA replication occurs at a rate of 50 nucleotides/second; at this rate, it would take almost two months to replicate the largest human chromosome. In reality, it takes only a few a hours because replication begins in many places at once. Eukaryotes are huge so need multiple origins of replication. The opening of the double helix at each origin of replication forms a replication bubble with a replication fork on either side.

Question 34

Why do we make use of the DNA replication method PCR?

Answer 34

To amplify areas of the genome we are interested in. PCR uses DNA primers and only needs one enzyme (taq polymerase). This is a special enzyme as it is very heat resistant so it survives the heat that breaks the DNA into two strands. It evolved to function in bacteria present in the hot springs of Yellowstone National park. DNA ligase is not needed as we are only synthesising short DNA fragments.

Question 35

What are chromosomes?

Answer 35

In most eukaryotes, there are 2 sets of chromosomes -> diploid. The sex chromosomes are X and Y (in mammals). Non-sex chromosomes are known as autosomal chromsomes. The human karyotype here shows 22 pairs of homologous chromosomes, numbered 1 - 22 from the longest to the shortest chromosome, and 1 pair of sex chromosomes.

Question 36

When are chromosomes in their condensed form?

Answer 36

When undergoing meiosis or mitosis. When uncondensed they occupy chromosome territories in the nucleus and are not entangled with one another but do share areas of interaction.

Question 37

What are homologous chromosomes?

Answer 37

In diploid organisms : one is inherited from each parent.

Question 38

What is a genetic locus?

Answer 38

It is the location of a particular gene on a chromosome. At each genetic locus, an individual has two alleles, one on each homologous chromosomes. Three gene pairs at three different loci - AA - homozygous - dominant, bb - homozygous - recessive, Cc - Heterozygous.

Question 39

What happens in chromosome replication?

Answer 39

Each strand of DNA starts with an RNA primer. On the leading strand : only one primer is required to start synthesis, replication continues to the end, the strand is replaced by DNA when the last fragment of the lagging strand at the opposite fork of the replication bubble catches up to it. On the lagging strand : multiple RNA primers are required, the final primer is added about 100 nucleotides from the 3' end of the template. When it is removed, the new daughter strand is shortened by about 100 nucleotides.

Question 40

Chromosome replication what do telomeres do?

Answer 40

Each end of a eukaryotic chromosome is capped by a repeating sequence called the telomere. In humans, the telomere concists of the sequence 5'-TTAGGG-3' repeated approximately 1500-3000 times. This end is shortened. In some cell types, before the next round begins, the enzyme telomerase replaces the missing nucleotides.

Question 41

Where is telomerase very active?

Answer 41

In germ cells (sperm and eggs) and also in stem cells (which are found in embryos and tissues that replenish cells with a high rate of turnover e.g. blood). Adult somatic cells (where telomerase is almost inactive) can only divide about 50 times before the telomeres become too short. This may contribute to ageing. In cancers, telomerase becomes active to allow uncontrolled growth and division

Question 42

What happens in cell division in prokaryotes?

Answer 42

During binary fission, a cell replicates its DNA, increases in size, and divides into two daughter cells. Each daughter cell receives one copy of the replicated parental DNA. In E. coli, the circular genome is attached by proteins to the inside of the plasma membrane. DNA replication is initiated at a specific location on the DNA molecule and proceeds in opposite directions around the circle. The cell then grows, as it elongates, the two DNA attachment sites move apart. When the cell is about twice its original size and the DNA molecules are well separated, a constriction forms at the midpoint of the cell. A new membrane and cell wall are synthesised at the site of the constriction, resulting in two daughter cells, each the same as the parent cell.

Question 43

What happens in cell division in eukaryotes?

Answer 43

Cell division in eukaryotes proceeds through a number of steps that make up the cell cycle. The cell cycle consists of two distinct phases - M phase : the time during which the parent cell divides into two daughter cells. Interphase : the time between two successive M phases.

Question 44

Cell division in Eukaryotes - What happens in the G1 phase?

Answer 44

Size and protein content of the cell increase in preparation for the S phase. During this phase, many regulatory proteins are made and activated.

Question 45

Cell division in Eukaryotes - What happens in the S phase?

Answer 45

Synthesis, in which the entire DNA content in the nucleus of the cell is replicated.

Question 46

Cell division in Eukaryotes - What happens in the G2 phase?

Answer 46

Cell prepares for mitosis and cytokinesis

Question 47

Cell division in Eukaryotes - What happens in the G0 phase?

Answer 47

Phase is present in cell types that do not actively divide. Cells in this phase would include liver cells, nerve cells, and those that form the lens of the eye.

Question 48

What needs to occur for cell division to proceed?

Answer 48

Every chromosome in the parent cell must be duplicated so that each daughter cell receives a full set of chromosomes. This duplication takes place during S phase. Even though the DNA in each chromosome is duplicated, the two identical copies, called sister chromatids, do not completely separate. They stay side by side, held together at the centromere.

Question 49

What is interphase?

Answer 49

Nuclear envelope visible, chromatin in the nucleus

Question 50

What are the 5 stages of Mitosis?

Answer 50

Prophase, Prometaphase, Metaphase, Anaphase, Telophase

Question 51

Mitosis - What happens in Prophase?

Answer 51

Chromosomes condense, centrosomes radiate microtubules and migrate to opposite poles. Prophase is characterised by the appearance of visible chromosomes. Outside the nucleus, the cell assembles the mitotic spindle, which is mostly made of microtubules that pull the chromosome apart into separate daughter cells. Centrosomes, the microtubule-organising centers for animal cells, are duplicated and begin to migrate to opposite poles. Plant cells also have microtubule-based mitotic spindles, but they lack centrosomes.

Question 52

Mitosis - What happens in Prometaphase?

Answer 52

During prometaphase, the nuclear membrane breaks down and the microtubules of the mitotic spindle attach to the chromosomes. The microtubules grow and shrink to explore the region that was once the nucleus. As the ends of the microtubules encounter chromosomes, they attach to the chromosomes at their centromeres.

Question 53

What are kinetochores?

Answer 53

Two protein complexes called kinetochores are associated with the centromere, one on each side of the centromere. Each kinetochore is associated with one of the two sister chromatids and forms the site of attachment for a single microtubule. This arrangement ensures that each sister chromatid is attached to a microtubule radiating from one of the poles of the cell.

Question 54

Mitosis - What happens in Metaphase?

Answer 54

Metaphase is one of the most visually distinctive stages of the cell during mitosis. During this stage, the chromosome are lined up in a single plane that is roughly equidistant from both of the spindle poles.

Question 55

Mitosis - What happens in Anaphase?

Answer 55

During anaphase, the sister chromatids are separate. The centromere holding a pair together divides, allowing the two sister chromatids to separate from each.

Question 56

Mitosis - What happens in Telophase?

Answer 56

During telophase, a complete set of chromosomes arrives at a spindle pole and cytosolic changes occur in preparation for the cell's division. The microtubules of the mitotic spindle break down and disappear, and the nuclear envelope begin to re-form around each set of chromosomes, creating two nuclei. Once the nuclear envelope is re-formed, the chromosomes decondense and becomes less visible. This stage marks the end of mitosis.

Question 57

What happens in cytokinesis?

Answer 57

As mitosis ends, cytokinesis begins and the parent cell divides into two daughter cells. In animal cells, this stage begins when a ring of actin filaments, called the contractile ring, forms against the inner face of the cell membrane at the equator of the cell perpendicular to the axis of what was the spindle. This ring contracts, pinching the cytoplasm of the cell and dividing it in two.

Question 58

What happens in cytokinesis in plants?

Answer 58

For the most part, mitosis is similar in animals and plant cells, but cytokinesis is different. Since plant cells have a new cell wall, the division of the cell is achieved by constructing a new cell wall. During telophase, the plant cells form a structure called the phragmoplast in the middle of the cell. The phragmoplast consists of overlapping microtubules that guide vesicles containing cell-wall components to the middle of the cell. During late anaphase and telophase, these vesicles fuse to form a new cell wall, called the cell plate, in the middle of the dividing cell. Once the cell plate is large enough, it fuses with the original cell wall at the perimeter of the cell and cytokinesis is complete.