DNA structure and replication Flashcards
why is hereditary material important to life?
- life depends on the ability of cells to store, retrieve and translate hereditary instructions required to make and maintain a living organism
- hereditary instructions are stored within every living cell as its genes, which determine the characteristics of a species as a whole and the individuals within it
what characteristics must a hereditary material possess?
- the hereditary material must have a high capacity for information storage and be chemically stable to be able to encode information without fail
- the hereditary material must replicate accurately
- the hereditary material must be capable of variation
what are the polymers and monomers of nucleic acids?
- nucleotides are monomers of nucleic acids that comprise polynucleotides
- polynucleotides are polymers of nucleic acids
what are the two types of nucleic acid and what are their characteristics?
- deoxyribonucleic acid (DNA), in which the pentose sugar is deoxyribose. deoxyribonucleotides are the monomers of DNA
- ribonucleic acid (RNA), in which the pentose sugar is ribose. ribonucleotides are the monomers of RNA
what is the chemical structure of pentose sugar?
pentose sugars are five-carbon sugars and occur as ring forms. in nucleic acids, the 5’ carbon is linked in an ester bond to the phosphate group and the 1’ carbon is linked in a glycosidic bond to the nitrogenous base
what is the main difference between deoxyribose and ribose sugars?
- at the 2’ carbon of deoxyribose, the OH group is replaced by a H atom
- the partial negative charge of the OH group in ribose repels the negative charge of the phosphate, preventing the RNA chain from coiling in as tight of a helix as it does in DNA
- hence, RNA is more susceptible to chemical and enzyme degradation
what is the chemical structure of nitrogenous bases?
a nitrogenous base has a nitrogen-containing ring structure. the nitrogenous bases fall into 2 types: purines and pyrimidines. purines have a 5-membered ring fused to a 5-membered ring, while pyrimidines have a 6-membered ring. adenine and guanine are purines, while cytosine, uracil and thymine are pyrimidines
what is the difference between thymine and uracil?
DNA contains thymine, while RNA contains uracil. thymine contains a methyl substituent at carbon-5, while uracil does not have a methyl substituent at carbon-5
what is the definition of a nucleoside? how does the formation of a nucleoside occur?
a nucleoside is a combination of a pentose with a nitrogenous base, which occurs with the elimination of water and therefore is a condensation reaction. 1’ carbon of the pentose is linked in a glycosidic bond to the nitrogenous base
how is a nucleotide formed?
a nucleotide is formed by further condensation between the nucleoside and phosphate group, forming a phosphodiester bond between the 5’ carbon of pentose and the phosphate group
how is a dinucleotide formed?
two nucleotides join to form a dinucleotide by condensation between the 5’ phosphate group of one nucleotide and the 3-hydroxyl group of the other to form a phosphodiester bond
how are polynucleotides formed?
the condensation reaction between nucleotides is repeated several millions of times to form a polynucleotide. phosphodiester bonds between 5’ phosphate and 3’ hydroxyl groups of nucleotides form a linear, unbranched sugar-phosphate backbone.
what is the function of phosphodiester bonds in a polynucleotide?
phosphodiester bonds are strong covalent bonds, and confer strength and stability on the polynucleotide chain, which prevents breakage of the chain during DNA replication
how are polynucleotides conferred polarity or directionality?
each DNA or RNA strand has two free ends that are chemically different from each other.
1. the 5’ end with a 5’ carbon carrying a phosphate group
2. the 3’ end with a 3’ carbon carrying a OH group
thus, every DNA or RNA molecule has an intrinsic polarity or directionality
what are the principles of nucleotide base composition of DNA, and complementary base pairing?
- the base composition of DNA of an organism is constant throughout all the somatic cells of that organism and is characteristic for a given species
- there is always an equal proportion of purines (A and G) and pyrimidines (C and T/U)
- adenine forms hydrogen bonds with thyme/uracil while guanine forms hydrogen bonds with cytosine
- the amount of adenine is always equal to the amount of thymine/uracil, while the amount of guanine is always equal to the amount of cytosine
what are the main features of DNA?
- DNA consists of two polynucleotide strands. each strand forms a right-handed helix, and both strands coil around each other to form a double-helix, with 10 base pairs every turn, so each turn has a length of 3.4nm
- the diameter of the helix is uniformly 2nm, leaving enough space of 1 purine and 1 pyramidine in the centre of the helix
- the strands are antiparallel, with one strand oriented in the 5’ to 3’ direction, while the other is oriented in the 3’ to 5’ direction
- each strand has a sugar phosphate backbone conferred strength due to phosphodiester linkages, with phosphate groups that project outside the double helix and nitrogenous bases that orientate inwards towards the central axis at almost right angles
- the bases of opposite strands are bonded together by relatively weak hydrogen bonds. 2 hydrogen bonds form between A and T, while 3 hydrogen bonds occur between C and G
- there are minor and major grooves between the sugar-phosphate backbones, which are large enough to allow protein molecules to gain access and make contact with the bases
why do phosphate groups project outwards, while nitrogenous bases orientate inwards towards the central axis?
- phosphate groups are hydrophilic and have an affinity for the surrounding aqueous medium
- nitrogenous bases are relatively hydrophobic and thus this puts them in the interior of the molecule and away from the surrounding aqueous medium
what is the significance of complementary base pairing?
- the base sequence in one strand determines the base sequences in the complementary strand. this is important in DNA replication and the transmission of the genetic information stored
- the weak hydrogen bonds make it relatively easy to separate the two strands of DNA. separating the AT pair is easier than separating the GC pair, since the AT pair involves 2 hydrogen bonds, and the GC pair involves 3 hydrogen bonds
why do nitrogenous bases form complementary base pairs?
- steric restrictions - the sugar phosphate backbone of each polynucleotide chain has a uniform diameter of 2nm. purines are about twice as wide as pyrimidines and thus they need to be paired to prevent steric hindrance
- hydrogen bond factors - each nitrogenous base has chemical side groups which have well defined positions, that can form either 2 or 3 hydrogen bonds with its appropriate partner.
what are the levels of DNA packing in a chromosome?
- DNA double helix
- beads on a string form of chromatin
- 30nm chromatin fibre of packed nucleosomes
- chromatin fibre loops along a central protein scaffold to form euchromatin and heterochromatin
- metaphase chromosome
what structural features stabilises the DNA double helix?
- extensive hydrogen bonds between base pairs
- hydrophobic interactions between the stacked base pairs
- exposure to outside influences of only the sugar-phosphate backbone
- nitrogenous bases being safely tucked inside the double helix
- DNA double-helix tightly wound around histones to form a repeating array of nucleosomes, which are eventually folded into higher order structures such as the chromosome.
what structural features results in invariant base sequence in DNA?
- specific, complementary base pairing between DNA strands, so that genetic information is present more than once in the DNA molecule
- if the base sequence in one of the two strands is accidentally altered, the cell discards the damaged strand. It then makes a perfectly good strand by using the remaining intact strand as a template
how does specific pairing of nitrogenous bases in DNA, aid in its function of DNA replication?
- the two strands of DNA are complementary, and each stores the information necessary to reconstruct the other
- when a cell copies a DNA molecule, each strand serves as a template for order nucleotides into a new, complementary strand
- there is now an exact replica of the ‘parent’ molecule to ensure faithful transmission of genetic instructions
what is watson and crick’s proposed model for DNA replication?
- the hydrogen bonds between complementary base pairs are broken and the 2 DNA strands unwind and separate from each other
- each DNA strand acts as a template for the assembly of a complementary strand
- nucleotides line up singly along the template DNA strand according to the rule of complementary base pairing
- DNA polymerases join the nucleotides together at their sugar-phosphate moieties
- this model is described as semi-conservative, where each of the two daughter DNA molecules consists of one parental DNA strand and one newly-synthesised daughter strand
what does the conservative model for DNA replication suggest?
the parental DNA molecule emerges from the replication process intact, and generates DNA copies consisting of entirely new molecules
what does the dispersive model for DNA replication suggest?
all four strands of DNA following replication, have a mixture of old and new DNA
what was the meselson-stahl experiment which tested for evidence for semi-conservative replication?
- for many generations, cells of E.coli were grown on medium containing only the heavy isotope of nitrogen. heavy nitrogen was incorporated into all the nitrogenous bases and the resulting DNA is known as heavy DNA
- this bacteria is then transferred to medium containing only the light isotope of nitrogen, and allowed to divide just once, producing the 1st generation of bacteria
- density-gradient centrifugal ion was performed on a DNA extract from the bacteria, and DNA was separated on the basis of density
- these bacteria were allowed to undergo a second round of replication and binary fission, producing the 2nd generation. DNA was again separated by density centrifugation
how does CsCl density-gradient centrifugation work?
- If CsCl solution is centrifuged at very high speeds for 48-72 hours, a CsCl density gradient with increasing density towards the bottom of the centrifugation tube is formed
- This is because of the equilibrium between sedimentation of the CsCl to the bottom of the spinning tube as a result of centrifugal forces, and diffusion of CsCl towards the top of the tube
- DNA molecules move to the position where their density equals that of CsCl and floats at that position
what were the results of the meselson-stahl experiment which tested for evidence for semi-conservative replication?
- in generation 0 of DNA, only heavy DNA was present
- in generation 1 of DNA, only hybrid DNA was present, which are intermediate in density, and contained one heavy and one light strand
- in generation 2 of DNA, hybrid DNA and light DNA was present in equal amounts
- these results can only be achieved if replication is semi-conservative
what are the characteristics of the semi-conservative model of DNA replication?
- extremely complex - the timing of steps is extremely precise, and the double helix must unwind while the replication machinery copies the two antiparallel strands simultaneously
- extremely fast - although each human cell has approximately 3 x 10^0 base pairs, it takes the cell just a few hours to copy all the DNA
- extremely accurate - the mutation rate is approximately 1 nucleotide change per 10^9 nucleotides each time the DNA is replicated
- requires the cooperation of a large team of enzymes and other proteins, as well as the expenditure of ATP
what are the steps of DNA replication?
- location of origins of replication
- separation of parental DNA strands
- synthesis of RNA primer
- synthesis of daughter DNA strands
what is the definition of origins of replication?
each origins of replication is a specific sequence of nucleotides, which is generally A-T rich. there are only 2 hydrogen bonds between each A-T base pair, hence it is easier to disrupt the bonds as less energy is required to overcome them
how is the DNA double helix separated?
- initiator proteins recognise this sequence and bind to the origins of replication sequence. the DNA double helix is separated into 2 strands, forming a replication bubble
- the length of DNA unwound to initiate replication is typically 50 base pairs, and ATP is required
- at the end of a replication bubble, a Y-shaped structure called a replication fork is synthesised. the two replication forks move away from the origin of replication proceed bidrectionally, until the entire DNA molecule is separated
what is the difference between separating DNA strands in prokaryotes and eukaryotes?
- circular DNA in prokaryotes only have a single origin of replication, while DNA double helix in eukaryotes have multiple origins of replication, hence multiple regions of the chromosome undergo replication simultaneously
- in prokaryotes, replication proceeeds biodirectionally to a termination site located approximately halfway around the circular chromosome, while in eukaryotes, replication proceeds bidirectionaly until replication bubbles fuse and synthesis of daughter strands is complete
how long does replication take in eukaryotes?
replication takes approximately 8 hours in human cells, but if it had only one origin of replication, it would take 100 times longer
why is there a continual need for the separation of the base pairs of the parental DNA molecule?
this is so that both DNA strands can act as templates for the synthesis of daughter DNA strands
what are the three proteins involved in the separation of parental DNA strands?
- helicases
- single-strand DNA binding proteins
- topoisomerases
what functions do helicases perform on DNA?
- after initiation, helicases bind to one strand of the DNA molecule
- using ATP, helicases break the hydrogen bonds holding the two strands of DNA together. this unwinds the DNA double helix and separates the parental DNA strands at the region of the replication fork
- each of the two parental DNA strands serve as the template for the synthesis of a new DNA strand
what functions do single-strand DNA binding proteins perform on DNA?
- the unwound single-stranded portion of the DNA double helix is temporarily stabilised by the binding of SSB proteins
- this keeps the two parental strands in the appropriate SS condition to act as template
- this protects the SS DNA, which is very unstable, from being degraded
how does single-strand DNA-binding proteins act on DNA?
- each SSB protein prefers to bind next to a previously bound molecule
- long rows of this protein form on a DNA single strand, and cooperative binding straightens out the DNA template and facilitates the DNA polymerisation process
what functions do topoisomers perform on DNA?
- unwinding causes tighter twisting and supercooling ahead of the replication fork, resulting in tension
- topoisomerases cleave a strand of the helix to create a transient SS nick, relieving strain on the DNA molecule by allowing free rotation around the intact strand
what are the two limitations of DNA polymerases?
- none of the DNA polymerases can initiate the synthesis of a DNA strand on its own. therefore, an RNA primer is used
- DNA polymerases only add dNTPs to the free 3’ end of a growing DNA strand, and never to the 5’ end so it can only elongate in the 5’ to 3’ direction. additionally, the two strands of a DNA double helix are antiparallel so continuous synthesis of both DNA strands at a replication fork is not possible
how is a RNA primer synthesised?
- a portion of the parental DNA strand serves as template for making the RNA primer with the complementary base sequence
- primase joins the ribonucleotides to make the primer, which is about 10 nucleotides long. hydrolysis of ATP is involved
what is the function of a RNA primer?
the RNA primer provides a free 3’ OH end that DNA polymerase can extend, thereby priming the synthesis of the daughter DNA strand. the DNA polymerase later replaces the RNA nucleotides of the primers with DNA versions
how are daughter DNA strands synthesised?
- DNA polymerase reads the template strand and assembles the dNTPs based on complementary base pairing
- when an incorrect base pair is recognised, DNA polymerase reverses its direction by one base pair of DNA, and the 3’ to 5’ exonuclease activity of the enzyme allows the incorrect base pair to be excised
- DNA polymerases catalyse phosphodiester bond formation between a growing daughter DNA strand and an incoming nucleotide
- the addition of a dNTP to the growing daughter DNA strand requires the formation of a phosphoester bond between the free 3’ hydroxyl group of the last nucleotide in the growing strand and the free 5’ phosphate group of the incoming dNTP
- the incoming dNTP loses a pyrophosphate group when they form the phosphoester bond with the growing daughter DNA strand. the energy released from pyrophosphate bond breakage is coupled to phosphoester bond formation
what is the definition of leading strand synthesis?
the complementary daughter DNA strand that is continuously synthesised as a single polymer along the template strand, is polymerised in the 5’ to 3’ manner towards the replication fork
what is the definition of lagging strand synthesis?
the complementary DNA strand that is discontinuously synthesised as a series of short fragments known as okazaki fragments, is polymerised in the mandatory 5’ to 3’ manner against the overall direction of the replication fork
how are okazaki fragments linked to produce a continuous DNA strand?
- each okazaki fragment requires an RNA primer for strand initiation
- DNA polymerase removes the RNA primer and replaces it with dNTPs
- DNA ligase then catalyses the formation of a phosphoester bond between the 3’ end of each new okazaki fragment and the 5’ end of the growing daughter DNA strand
what is the end replication problem in linear chromosomes?
- each time a cell with linear chromosome divides, a small section at the extreme 3’ end of the parental strand does not undergo DNA replication
- this is because when the final RNA primer at the end of lagging strand is removed, there is no upstream strand onto which DNA polymerase can build to fill the resulting gap
- hence, the daughter DNA strand resulting from lagging strand synthesis would be shortened with each round of replication, and this leads to the shortening of telomeres in chromosomes
