molecular biology of the cell chapter 5 summary PDF

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Chapter 5: DNA replication, repair and recombination underlies DNA replication and DNA repair. The DNA double helix acts as a template for its own duplication. The two daughter cells are exact copies of the parent cells. This is called semi conservative replication. Nucleotides are the building bloc...

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Chapter 5: DNA replication, repair and recombination Base-pairing underlies DNA replication and DNA repair. The DNA double helix acts as a template for its own duplication. The two daughter cells are exact copies of the parent cells. This is called semi conservative replication. Nucleotides are the building blocks and have to be in the ATP form. The chain grows from the 5’ to the 3’ end. It cannot grow the other way. On the picture on the right the chemistry of DNA synthesis is shown. The addition of a deoxyribonucleotide to the 3’ end of a polynucleotide chain (primer strand) is the fundamental reaction by which DNA is synthesised. Base pairing between an incoming deoxyribonucleoside triphosphate and an existing strand of DNA (template strand) guides the formation of the new strand of DNA and causes it to have a complementary nucleotide sequence. An enzyme is needed for DNA replication, called DNA polymerase. It catalyses DNA synthesis. DNA polymerase can open the DNA and maintain the opening and it can add new nucleotides. The DNA polymerase upon incorporation releases triphosphate. This is done nucleotide by nucleotide and this can be up to 500 nucleotides per second. DNA polymerase catalyses the stepwise addition of a deoxyribonucleotide to the 3’-OH end of a polynucleotide chain, the growing primer strand that is paired to an existing template strand. The newly synthesised DNA strand therefore polymerises in the 5’-to-3’ direction. Because each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognised by the DNA polymerase, this strand determines which of the four possible deoxyribonucleotides (A,C,G or T) will be added. The reaction is driven by a large, favourable free-energy change, caused by the release of pyrophosphate and its subsequent hydrolysis to two molecules of inorganic phosphate. A schematic diagram of DNA polymerase is shown on the right. The proper base-pair geometry of a correct incoming deoxyribonucleoside triphosphate causes the polymerase to tighten around the base pair, thereby initiating the nucleotide addition reaction. Dissociation of pyrophosphate relaxes the polymerase, allowing translocation of the DNA by one nucleotide so the active site of the polymerase is ready to receive the next deoxyribonucleoside triphosphate. The DNA replication fork is asymmetrical. The structure of the replication fork is shown on the picture on the right. The left picture shows the DNA synthesises on the lagging strand must be made initially as a series of short DNA molecules. These are called Okazaki fragments. The right picture shows the same fork a short time later. On the lagging strand, the Okazaki fragments are synthesised sequentially, with those nearest to the fork being the most recently made. The high fidelity of DNA replication requires several proofreading mechanisms. Exonucleolytic proofreading of DNA polymerase during DNA replication: 1. 2. 3.

1 C is accidentally incorporated at the growing 3’-OH end of a DNA chain. The part of DNA polymerase that removes the mis incorporated nucleotide is a specialised member of a large class of enzymes called exonucleases. Exonucleases cleave nucleotides one at a time from the ends of polynucleotides.

The cell has a system that will recognise the wrong nucleotide, as stated above. It will start again and fill in with the right nucleotide. This is thanks to the 3’ to 5’ exonuclease activity attached to DNA polymerase. Cytosine has a tautomer which is an imino. The imino has lost a hydrogen. Instead of three hydrogen bonds this will have two hydrogen bonds, so it can pair with an A. That is why there can be a mistake in DNA replication. The 3’ to 5’

growing will never work, because it is not possible to have proofreading. This is because the high energy bond is on the wrong side and therefore will not be cleaved. There will be too many mistakes as a result because no proofreading can be done. Editing by DNA polymerase happens at the E-side. DNA polymerising happens at the P-side. If there is a mistake, the strand goes to the E side and the wrong nucleotide is taken out there. In human cells that are being replicated, there are .6 mutations happening. A certain level of mutations is needed to be able to adapt to a changing environment. A special nucleotide-polymerising enzyme synthesises short RNA primer molecules on the lagging strand. The reaction is catalysed by DNA primase, the enzyme that synthesises the short RNA primers made on the lagging strand using DNA as a template. Unlike DNA polymerase, this enzyme can start a new polynucleotide chain by joining two nucleoside triphosphates together. The primase synthesises a short polynucleotide in the 5’-3’ direction and then stops, making the 3’ end of this primer available for the DNA polymerase. The 3’-OH group is essential for RNA primer. The DNA polymerase can erase the RNA primer. The ligase has to fill the gap where the RNA primer is removed. The synthesis of one of many DNA fragments on the laggings strand is shown on the picture below. It costs a lot of energy to replicate the lagging strand, because there has to be an RNA primer used to keep starting the synthesis. DNA primase synthesises new RNA primers. DNA ligase is an enzyme that needs energy to fill up the gap in the DNA strands. This DNA ligase is nowadays known for a different reason. It has been the enzyme to do recombinant DNA. They could combine DNA from different organisms together because of DNA ligase.

DNA ligase seals a broken phosphodiester bond. DNA ligase uses a molecule of ATP to activate the 5’ end of the nick before forming the new bond. In this way, the energetically unfavourable nick-sealing reaction is driven by being coupled to the energetically favourable process of ATP hydrolysis. Special proteins help to open up the DNA double helix in front of the replication fork. DNA helicase opens up the double strand. It runs ahead of the opening, so the double strand DNA can open into one stranded DNA. This process uses energy from ATP hydrolysis. Single-strand DNA binding proteins (SBB proteins) have an effect on the structure of a single-strand DNA. Because each protein molecule prefers to bind next to a previously bound molecule, long rows of this protein form on a DNA single strand. This cooperative binding of SBB proteins straightens out the DNA template and facilitates the DNA polymerisation process. The hairpin helices, which are single-strand DNA, result from a chance matching of short regions of complementary nucleotide sequence; they are all similar to the short helices that typically form in RNA molecules. A sliding ring holds a moving DNA polymerase onto the DNA: 1. The structure of the clamp loader resembles a screw nut, with its threads matching the grooves of double-stranded DNA. 2. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. 3. The clamp loader, thanks to its screw-nut structure, recognises the region of DNA that is double -stranded and latches onto it, tightening around the complex of a template strand with a freshly synthesised elongating strand. 4. It carries the clamp along this double-stranded region until it encounters the 3’ end of a primer, at which point the loader hydrolyses ATP and releases the clamp, allowing it to close around the DNA and bind to DNA polymerase. The proteins at a replication fork cooperate to form a replication machine. A bacterial replication fork: o The lagging-strand DNA is folded to bring the laggingstrand DNA polymerase molecule into a complex with the Okazaki fragment. o Because the lagging-strand DNA polymerise molecule remains bound to the rest of the replication proteins, it can be reused to synthesise successive Okazaki fragments. o In the diagram, it is about to let go of its completed DNA fragments and move to the RNA primer that is just being synthesised. A winding problem arises during DNA replication: o DNA topoisomerases prevent DNA tangling during replication. o The replication fork cannot roll, therefor it is too big. o By rotating torsion is build up. This has to be relieved. o The cell cuts one of the strands, so the topoisomerases take away the torsion on DNA strands. o DNA topoisomerases cuts the strand and later attaches it together again.

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DNA topoisomerase plays a huge part in the fast replication of DNA.

The reversible DNA nicking reaction catalysed by a eukaryotic DNA topoisomerase 1 enzyme is shown in the pictures on the right. One end of the DNA double helix cannot rotate relative to the other end, this is fixed by type 1 DNA topoisomerase with tyrosine at the active site: o DNA topoisomerase covalently attaches to a DNA phosphate, thereby breaking the phosphodiester linkage in one DNA strand. o The two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain. o The original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reacting reversible. o Spontaneous re-formation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase. The DNA-helix-passing reaction catalysed by DNA topoisomerase is shown on the pictures on the right. DNA topoisomerase 2 can cut two strands instead of one. Two circular DNA double helices that are interlocked: o Topoisomerase 2 recognises the entanglement and makes a reversible covalent attachment to the two opposite strands of one of the double helices (orange) creating a double strand break and forming a protein gate. o The topoisomerase 2 gate opens to let the second DNA helix pass. o The gate shuts, releasing the red helix. o Reversal of the covalent attachment of topoisomerase 2 restores an intact orange double helix. o This gives two circular DNA double helices that are separated If there are fast dividing cells, it means the topoisomerase has to work efficiently. Podophyllotoxin is a compound that blocks topoisomerase. It is found in ‘fluitenkruid’ and it is tried to turn it into a medicine against cancer by turning it into etoposide, which is used to fight cancer. This is a powerful inhibitor of topoisomerase type 2. It is a cytostatic. It has different sugars attached than podophyllotoxin from ‘fluitenkruid’. The tumour cells cannot replicate so they die.

DNA replication is fundamentally similar in eukaryotes and bacteria. DNA synthesis begins at replication origins. A replication bubble (last step) formed by replication-fork initiation is shown on the right. In bacteria there is two replication forks. There is a leading and lagging strand DNA in bacteria too. o Local opening of DNA helix. o RNA primer synthesis. o Leading-strand DNA synthesis begins. o RNA primers start lagging-strand synthesis.

Bacterial chromosomes typically have a single origin of DNA replication. The replication begins at the replication origin. At the end of the DNA replication of a bacterial genome two circular daughter DNA molecules are present. A eukaryote has chromosomes. They are not circular, but linear. Prokaryotes are bacteria. Eukaryotic chromosomes contain multiple origins of replication. The structure of a portion of telomerase is shown on the right. Telomerase replicates the ends of chromosomes. It is a large protein-RNA complex (blue). The RNA contains a templating sequence for synthesising new DNA telomere repeats. The reaction itself is carried out by the reverse transcriptase domain of the protein (green). A reverse transcriptase is a special form of polymerase enzyme that uses an RNA template to make a DNA strand; telomerase is unique in carrying its own RNA template with it. Telomerase also has several addition protein domains that are needed to assemble the enzyme at the end of chromosomes. Telomere replication is shown on the right. Reactions that synthesise the repeating sequences that form the ends of the chromosomes (telomeres) o The 3’ end of the parental DNA strand is extended by RNA-templated DNA synthesis. o This allows the incomplete daughter DNA strand that is paired with it to be extended in its 5’ direction. o This incomplete, lagging strand is presumed to be completed by DNA polymerase alfa, which carries a DNA primase as one of its subunits. Telomerase fixes the problem of chromosome ends not being replicated properly by adding to the parenting strand, so the primer can start the last part of copying the lagging strand. Telomeres are packaged into specialised structures that protect the ends of chromosomes. Healthy chromosomes have a t-loop. Telomere length is regulated by cells and organisms. Chromosome shortening is known for ageing. The cells lose the ends of the chromosomes, so they lose genetic data. This is fixed by telomerase. Single point mutation can cause very serious damage. Chemical modifications of nucleotides produce mutations. Without DNA repair, spontaneous DNA damage would rapidly change DNA sequences. o If there is a deaminated C, 50% of the cells that come from the deaminated C have been mutated. o A depurinated A makes the loss of a base pair happen. Also 50% of the cells will be mutated after DNA replications.

A strand-directed mismatch repair system removes replication errors that escape from the replication machine. The MutS system is a proofreading system that can detect an error. The MutL can cut the DNA and remove the strands. The gap will be repaired by DNA synthesis. Mutation in MutS result in hereditary form of colon cancer, because they cannot repair the mutations efficiently enough. Many mutations are associated with diseases. DNA damages can be removed by more than one pathway, and excision repair is one of the repair systems....