4 - DNA Structure and Function Flashcards
Central dogma of molecular biology
Pictorial Representation of DNA
- Please note :
• The 5’‐ End
• The 3’‐ End
• The 3’ to 5 ‘ –Phosphodiester bond - Shorthand notation
- Extreme shorthand notation
- Very extreme shorthand notation
- Fig. 29.2 D is read, “ Thymine, adenine, cytosine, guanine”
- The phosphodiester linkages between nucleotides can be hydrolyzed by different nucleases (deoxyribonucleases for DNA, and ribonucleases for RNA)
Axis of symmetry
- The chains are paired in an antiparallel manner
- The hydrophilic deoxyribose – phosphate backbone of each chain is on the outside of the molecule; the hydrophobic bases are stacked inside.
- Two grooves are created: a major and a minor groove
- Base pairs run perpendicular to the helical axis
Complementary DNA Sequences
- The bases of one strand are always paired with the bases of the second strand
- Adenine is paired with thymine, and Guanine is paired with cytosine
- Chargaff’s Rule states that the amount of adenine equals thymine, and that the amount of guanine equals cytosine. The amount of purines equals pyrimidines
In-Class Worksheet 1
In-Class Worksheet 2
In-Class Worksheet 3
Class Notes
- Polymorphism = genes come in many varieties
- Nature has many polymorphisms
- Gene function depends on environment
- Expressivity = how likely a gene is expressed in the individual
- ex: high expressivity of breast cancer gene, have massectomy. low expressivity make lifestyle changes
- Much variation so it is difficult to predict
- Many methods of increasing genetic randomness
- Asxual is more efficient but less random
- Sexual introduces variations (diversity)
- Diversity is essential for evolution
- Examples of reproductive randomness
- Females are XX, only one X is activated at random
- Random sperm fertilizes the egg
- Females have 2 million eggs, one ovulated is random
- Biological determinationism = idea that genes determine everything about you
- The basis of natzism, all racism, that genes dictate what you are so you must be killed
DNA Structure
- There are two hydrogen bonds between A and
T, and there are three hydrogen bonds between G
and C. (The structure of the double helix is also
stabilized by the hydrophobic interactions
between the stacked bases.)
• 2. The two strands can be separated by heat or by
chemical treatment ( ionization of the nucleotide
bases).
• 3. The loss of helical structure is known as
denaturation, and can be monitored by
measuring the absorbance at 260 nm.
Single stranded DNA increases absorbance vs double stranded DNA
Denature with increased heat and decreased salt
Renature with decreased heat and increased salt
Salt keeps DNA renatured
Forms of DNA
• There are three major structural forms of DNA:
• 1. B form…Major form of chromosomal DNA, is a righthanded
helix with 10 base pairs per 360 degrees turn.
• 2. A form… Found in DNA‐RNA and in double‐stranded
RNA, is formed by moderate dehydration of the B form.
It is a right hand helix with 11 base pairs per 360
degrees turn.
• 3. Z form…Found in regions of DNA that contain
alternating purines and pyrimidines, the deoxyribosephosphate
backbone zigzags (Why do you think it is
called “Z” ?). Is a left‐handed helix that contains 12
base pairs per turn.
Linear and Circular DNA
- Nuclear eukaryotic DNA consists of long, linear
molecules of double‐stranded DNA called chromosomes - Mitochondrial DNA is circular. Because of its heirtage
- The prokaryotic genome usually consists of a single,
circular, supercoiled, double‐stranded molecule of
DNA. The chromosome is associated with nonhistone
proteins to form the nucleoid - Most species of bacteria contain small, circular
extrachromosomal DNA molecules known as plasmids
DNA Replication
• DNA replicates in a semiconservative manner.
• Each strand can serve as a template for the
synthesis of a complementary strand, oriented
in an antiparallel manner.
Replication of the DNA in a prokaryote entails the
following:
1. Localized “melting”, or opening of the double helix at
the origin of replication, a single, unique nucleotide
sequence that includes short AT –rich segments
(WHY?)
2. The unwinding and separation of the two strands, as
synthesis occurs at two replication forks that move
away from the origin in opposite directions. This
generates a replication bubble.
3 The continuation of bidirectional replication.
4. DNA synthesis proceeds in a 5’ to 3’ direction.
Proteins Required for Prokaryotic
Replication
- Replication requires the following:
- DNA Polymerase III
- Serves as the major enzyme for the synthesis of DNA during replication
- Is highly processive = it proceeds along the parental strand
- Requires dATP, dTTP, dCTP, and dGTP
- Enzyme removes a pyrophosphate from the 5’‐ deoxyribonucleotide triphosphates and attaches the new deoxynucleoside monophosphate to the growing chain. Losing PPi drives the reaction forward.
- Where have you seen the production of PPi, with its subsequent hydrolysis to 2 Pi ? glycogen and dUTP
- DNA Polymerase I
- Serves to fill the gaps between segments of DNA made by Pol. III
- DNA Polymerase II
- Serves role in repair of DNA
- DNA Polymerase III
Accessory Proteins
Other proteins required for Prokaryotic DNA synthesis include:
- DnaA protein
- Enhances melting in origin of replication
- Melting requires ATP (as energy source)
- DNA helicases
- Unwind the double helix (causing supercoiling in other regions of the DNA)
- Require ATP
- Single‐stranded DNA‐binding protein
- Bind to the single‐stranded DNA generated by helicases
- Binding exhibits cooperativity ( Discuss) Similar to hemoglobin
- Keep the two strands in the region of the replication origin separated
- Helps protect ssDNA from nulceases in the cell
- Type I DNA topoisomerases
- Can relax either or both negative and/or positive supercoils
- Enzymes function by reversibly cleaving one strand of the double helix and passing the intact strand through the break before it is resealed
- Type II DNA topoisomerases
- Enzymes bind tightly to dsDNA and reversibly creates nicks in both strands and passing a second stretch of the DNA to pass
through the break to reseal the break. - Enzymes can relieve both negative and positive superccoils
- DNA gyrase is a type II topoisomerase, which is involved in both DNA replication and also in transcription (Explain)
- Enzymes bind tightly to dsDNA and reversibly creates nicks in both strands and passing a second stretch of the DNA to pass
Clinical Correlation
• 1. There are anticancer agents that target
both human type I and type II topoisomerases
• 2. There are antimicrobial agents that target
bacterial DNA gyrase
Both prevent DNA replication
Direction of DNA Replication
- The DNA polymerases can read the parental strand in the 3’ to 5’ direction, meaning that they synthesize the new strand in the antiparallel 5’ to 3’ direction.
- The two newly synthesized DNA chains are synthesized as a leading strand, which is synthesized continuously, and also a lagging strand, which is synthesized discontinuously.
- Synthesis of the lagging strand entails the sequential actions of enzymes that initiate, elongate, and subsequently ligate fragments of DNA, each about 1000 nucleotides in length.
- Theses fragments are known as Okazaki fragments
Elongation of Leading and Lagging Strands
Priming
- DNA polymerases require the presence of an RNA primer.
- The RNA primer consists of a short, double‐stranded region of RNA base‐paired to the DNA template. This RNA primer has a free hydroxyl group on the 3’terminus, which accepts a deoxynucleotide through the catalytic agency of a DNA polymerase.
(Where did you see a primer beforehand ?) glycogen construction - Primase (which is a specific RNA polymerase) is responsible for synthesizing the short RNA sequences that are both complementary and antiparallel to the DNA template.
- Which strand has more RNA primers, the leading or the lagging strand ? Lagging
Chain elongation
- The genome of E.coli consists of about 4.7 million base pairs. DNA replication proceeds at a speed of about 1,000 nucleotides per second. ( Which means that it is carried out in about 40 minutes).
- Which enzyme is responsible for elongation ? DNA pol III
- What other function does this enzyme carry out, and why ?
Fidelity of Replication
- Newly replicated DNA has about one error per every 10 million nucleotides. How many mutations are introduced in the replication of a single E. coli genome ? 2 or 3 errors is high fidelity. Very few errors
- What does this mean ? E. coli can replicate faster than humans, but eukaryotes have multiple origins of replication and therefore quickens the process
- Proofreading function of **exonuclease **excises mismatched nucleotides
Removal of RNA Primer and Filling Gaps
- Removal of RNA primer and the filling of the gaps by DNA Polymerase I
- RNA primer is elongated by DMA polymerase III until another stretch of RNA is encountered.
- RNA primer is excised by DNA polymerase I one ribonucleotide at a time.
- Gap is filled by DNA polymerase I
- Remaining nick is sealed by DNA ligase
Eukaryotic DNA Replication
Both eukaryotic and prokaryotic DNA
replication are characterized by:
1. Bi‐directional processes
2. Necessity for primers
3. DNA polymerases synthesize in a 5’ to 3’ direction
4. Having leading and lagging strands
However…
Eukaryotic DNA has:
1. Linear, not circular, chromosomes
2. Much more packaging (nucleosomes, scaffolds, and higher order packing)
3. Much more genetic material (The human genome is over 600 times greater than that of E.coli)
4. Slower acting DNA polymerases
How are these issues resolved ? Multiple origins of replicaiton
There are a number of different eukaryotic DNA polymerases: DNA Polymerases alpha through epsilon
- What are the criteria for developing these five major categories of DNA Polymerase ? size, location in cell, inhibition, substrates
- Know: Polymerase Alpha = contains primase, initiates DNA synthesis, no proofreading
Quiescent Cells
-
Quiescent cells (cells that have ceased to
divide) can be stimulated to re‐enter the G1
phase.- The cell cycle is controlled by a series of
checkpoints between the phases -
Cyclins and cyclin‐dependent kinases
comprise important regulatory cell cycle
proteins
- The cell cycle is controlled by a series of
Telomeres
- Telomeres consist of noncoding DNA complexed with protein (shelterin) at the ends of the chromosome. Telomeres maintain the structural integrity of the chromosome and minimize nuclease attack.
- Human telomeric DNA consists of several thousand
tandem repeats of a noncoding hexameric sequence
AGGGTT, which are base‐paired to its complement. - Telomeres generally shorten with each round of cell
division, because after removal of the RNA primer from the extreme 5’‐ end of the lagging strand, the remaining gap cannot be filled in with DNA. - Once telomeres are shortened beyond a critical length, the cell is no longer capable of division.
