2.7 - 7.1: DNA replication Flashcards
Explain the start of DNA replication in eukaryotes
- DNA replication is initiated at many points in eukaryotes
- This makes the process of replication faster and more efficient
- The points are called the origins of initiation and will have very similar or the same base sequence
- proteins called origin recognition complexes will bind here and allow DNA helicase to attach, thus beginning the process of replication.
Summary of enzymes involved in DNA replication
- DNA gyrase moves in advance of helicase and relieves strain and prevents supercoiling on the separated strands.
- DNA helicase unwinds and separates the double stranded DNA by breaking the hydrogen bonds between base pairs.
- DNA polymerase 3 adds deoxynucleoside triphosphates to the 3’ end of the polynucleotide chain, synthesising in a 5’ - 3’ direction.
- RNA primase lays down an RNA primer on each template strand to provide an attachment and initiation point for DNA polymerase 3
- DNA polymerase 1 removes the RNA primers and replaces them with DNA
- DNA ligase joins the okazaki fragments together to create a continuous strand.
What does DNA helicase do
- Unwinds the DNA helix
- separates the two polynucleotide strands by breaking the hydrogen bonds between complementary base pairs
- ATP is needed by helicase to both move along the DNA molecule and to break the hydrogen bonds
- The two separated strands become parent/template strands for the replication process.
Describe how DNA polymerase is used, and what RNA primers are
- RNA primers laid by RNA primase provide and attachment and initiation point for DNA Polymerase 3
- RNA primers consists of a short sequence (10 base pairs) of RNA nucleotides
- DNA polymerase 3 always moves in a 5’ to 3’ direction
- DNA polymerase 3 catalyses the covalent phosphodiester bonds between sugars and phosphate groups of nucleotides
- DNA polymerase 3 adds new nucleotides to the C 3’ hydroxyl group in the sugar of a nucleotide
- DNA polymerase proof reads the complementary base pairing. Consequently mistakes are very infrequent occurring approximately once in every billion base pairs
Describe where energy is gotten and what nucleotides really are
- free nucleotides are deoxynucleoside triphosphate
- The extra phosphate groups carry energy which is used for formation of covalent bonds
The replication of DNA is semi conservative and depends on complementary base pairing
- Each of the nitrogenous bases can only pair with its partner (A=T G=C) This is called complementary base pairing
- The two new strands formed will be identical to the original strand
- Each new strand contains one original and one new strand, therefore DNA replication is said to be a semi-conservative process
Detailed summary of DNA Replication
- DNA replication occurs during (S phase of ) interphase, in preparation for cell division
- Helicase unwinds the double helix separating the strands of DNA
- It breaks the hydrogen bonds between the two strands
- Single stranded binding proteins keep the separated strands apart so that nucleotides can bind
- DNA gyrase moves in advance of helicase and relieves strain and prevents the DNA supercoiling again.
- each strand of parent DNA is used as template for the synthesis of the new strands
- synthesis always occurs in 5´ → 3´ direction on each new strand
- Therefore synthesis is continuous on leading strand (in the same direction as helicase) and dis-continuous on lagging strand (away from from helicase)
- This leads to the formation of Okazaki fragments on the lagging strand
- To synthesise a new strand first an RNA primer is synthesized on the parent DNA using RNA primase
- Next DNA polymerase III adds the nucleotides (to the 3´ end) added according to the complementary base pairing rules; adenine pairs with thymine and cytosine pairs with guanine; (names needed, letters alone not accepted)
- Nucleotides added are in the form of as deoxynucleoside triphosphate. Two phosphate groups are released from each nucleotide and the energy is used to join the nucleotides in to a growing DNA chain.
- DNA polymerase I then removes the RNA primers and replaces them with DNA
- DNA ligase next joins Okazaki fragments on the lagging strand
- Because each new DNA molecule contains both a parent and newly synthesised strand DNA replication is said to be semi-conservative.
Polymerase Chain Reaction (PCR)
- Typically used to copy a segment of DNA – not a whole genome
- Used to amplify small samples of DNA In order to use them for DNA profiling, recombination, species identification or other research.
- The process needs a thermal cycler, primers, free DNA nucleotides and DNA polymerase.
To summarize:
PCR is a way of producing large quantities of a specific target sequence of DNA. It is useful when only a small amount of DNA is available for testing e.g. crime scene samples of blood, semen, tissue, hair, etc.
PCR occurs in a thermal cycler and involves a repeat procedure of 3 steps:
- Denaturation: DNA sample is heated to separate it into two strands
- Annealing: DNA primers attach to opposite ends of the target sequence
- Elongation: A heat-tolerant DNA polymerase (Taq) copies the strands
One cycle of PCR yields two identical copies of the DNA sequence
- A standard reaction of 30 cycles would yield 2^30 copies of DNA
What experiment supported the theory of semi-conservative DNA replication
- Before Meselson and Stahl’s work there were different proposed models for DNA replication. (dispersive, conservative) After their work only semi-conservative replication was found to be biologically significant.
1) DNA from bacteria that had been grown in medium containing 15N appeared as a single band
2) DNA is transferred to the N14 medium to replicate
3) After one round of replication, the DNA appeared as a single band intermediate between that expected for DNA with 15N and that expected for DNA with 14 N.
4) After a second round of replication in the 14 N medium, DNA appeared as two bands, one in the position of hybrid DNA (half 15N half 14N) and the other in the position of DNA that contained only 14N.
Some regions of DNA do not code for proteins but have other important functions. List the name of the regions and their functions.
The percentage varies greatly between organisms, but in all organisms there are regions of DNA that are not expressed as polypeptides. This non-coding DNA is still important to organisms for a variety of reasons.
Genes, the regions of DNA that code for polypeptides, contain both intron and exon DNA.
- Introns are ‘edited’ out of messenger RNA (mRNA)
- mRNA is translated by ribosomes into polypeptides
- Therefore only exons code for the polypeptides
Between genes exist non-coding regions of DNA. Although such DNA does not code for polypeptides it can affect transcription of mRNA.
- Promoters sequences are attachment points for RNA polymerase adjacent to the gene
Some of these regions act as binding sites for particular proteins, which in turn affect transcription of the nearby gene:
- Enhancers are sequences that increase the rate of transcription (when a protein is bound to it)
- Silencers inhibit transcription (when a protein is bound to it)
- The end of chromosomes contain highly repetitive DNA sequences.
- These regions are called Telomeres and they protect the DNA molecule from degradation during replication.
Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing.
Dideoxyribonucleic acid stops DNA replication when it is added to a new DNA strand.
Fluorescent dye markers are attached to dideoxyribonucleic acids so that the base present when replication stops can be identified. From this the base on the parent strand deduced.
DNA replication is carried out with dideoxyribonucleic acid mixed in with normal deoxyribonucleic acid.
A range of new strands of differing lengths are produced.
The length of strand and the terminal base are identified by sequencing machines.
Tandem repeats
Tandem repeat sequences are short sequences of (non-coding) DNA, normally of length 2-5 base pairs, that are repeated numerous times in a head-tail manner.
How tandem repeats are used in DNA profiling?
- The TRs vary greatly in terms of the different number of copies of the repeat element that can occur in a population.
- For maternal profiling it is usually mitochondrial DNA.
- For paternal profiling commonly the Y chromosome is used.
- Chromosomes occur in pairs (if not using the Y chromosome) and the tandem repeat on each may vary.
- Dyes markers (e.g. attached to dideoxyribonucleic acids) are attached to the tandem repeats during PCR (DNA Replication).
- Restriction enzymes can be used to cut DNA between the tandem repeats.
- Electrophoresis enables scientists therefore to calculate the length of the tandem repeat sequence of an individuals.
- If different tandem repeats at different loci are used then a unique profile, for an individual can be identified.
Electrophoresis
technique to separate DNA fragments according to their size
