2. DNA Replication and Repair - Essential Cell Biology PDF

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DNA REPLICATION AND REPAIR DNA REPLICATION 

When it comes to replication, each strand of DNA serves as a template for the synthesis of a new complementary strand. For example, S strand can serve for making a new strand S’, while strand S’ can serve as a template for making new strand S. DNA replication produces to complete double houses from the original DNA molecule. Each new DNA helix being identical in nucleotide sequence to original DNA double helix. Each of the daughter DNA ends up with one of the original strands plus one completely new; this style of application is said to be semiconservative.

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DNA strands are locked together by a large numbers of hydrogen bonds. To be used as a template, the double helix must be first opened up and the two strands separated to expose unpaired bases. The process of DNA synthesis is begun by initiator proteins that bind to DNA sequences called replication origins. Here the initiator pry DNA strands apart breaking hydrogen bonds. Once an initiator protein binds to DNA, it attracts a group of protein that carry out DNA replication.

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DNA molecules in the process of being replicated contain Y-shaped junctions called replication forks, formed at each replication origin. DNA replication in bacteria and eukaryotic chromosomes is termed bidirectional and the forks move at about 1000 nucleotide pairs per second; in humans about 100 nucleotide pairs per second.

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The movement of a replication fork is driven by DNA polymerase. This enzyme catalyses the addition of nucleotides to the 3’ end of a growing DNA strand, using parental DNA as a template. The final product is a new strand of DNA that is complementary in nucleotide sequence to the template. Polymerization reaction involves formation of a phosphodiester bond between the 3’ end and the 5’-phosphate group of the incoming nucleotide, which enters the reaction as a deoxyribonucleoside triphosphate. Energy is provided by deoxyribonucleoside triphosphate itself.

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Each strand of a DNA double helix has a unique chemical direction determined by the way each sugar residue is linked to the next, the two strands in the double helices are antiparallel. At each replication fork, one new DNA strand is being made on a template that runs in one direction (3’ to 5’), while the other new strand is being made on a template that runs in the opposite direction (5’ to 3’). The replication fork is therefore asymmetrical. DNA polymerases adds subunits only to the 3’ end of a DNA strand. A new DNA chain can be synthetized only in a 5’-to-3’ direction. DNA strand that appears to grow in the incorrect direction is made discontinuously, with DNA polymerases moving backward with respect to the direction of replication for movement so that each new DNA fragment can be polymerised in the 5’-to-3’ direction. The resulting small DNA pieces (Okazaki fragments) are joined together to form a continuous new strand (lagging strand); the leading strand is instead the continuously synthesised one.

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DNA polymerase makes only one error every 107 nucleotide pairs it copies. If incorrect base pairs are allowed to remain, they will result in an accumulation of mutations. This is avoided because the enzyme has special qualities which increase accuracy of DNA replication that carefully monitors the base-pairing between incoming nucleotide and the template strand. When DNA polymerase makes a mistake and adds the wrong nucleotide, the error can be corrected through proofreading. Before the enzyme adds the next nucleotide to a growing DNA strand, it checks whether the previously added nucleotide is correctly base-paired. If not, polymerase clips off the mispaired nucleotide and tries again. Proofreading is carried out by nuclease that cleaves the phosphodiester backbone.

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Accuracy of DNA replication depends on the requirements of the DNA polymerase for correctly base-paired 3’ end before it can add more nucleotides to a growing DNA strand. Primase is an example of an RNA polymerase, an enzyme that synthesises RNA using DNA as a template. Chemically speaking, RNA and DNA are similar but an RNA primer is synthesised on dying by complementary base paving in exactly the same way as is DNA. Primase can start a new polynucleotide chain by joining together to nucleoside triphosphates without the need for a base-paired 3’ end as a starting point.

A nuclease degrades the RNA primer, a DNA polymerase called repair polymerase replaces the RNA with DNA. The enzyme DNA ligase joins the 5’-phosphate end of one DNA fragment to the 3’-hydroxyl end of the next. Primers frequently contain mistakes but stand out and “suspect copy” to be automatically removed and replaced by DNA. 

For DNA replication to occur, the double helix must be unzipped ahead of the replication fork, as the incoming nucleoside triphosphate can form base pairs with each template strand. DNA helicases and single-strand DNA-binding proteins cooperate. The first sits at the very front of the replication machine, the second cling to the single-stranded DNA exposed by the helicase preventing the strands to re-form base pairs and keeping them elongated so that they can serve as templates. As DNA helicase unwinds DNA double helix, it generates a section of overwound (supercharged) DNA. Tension builds up because the chromosome is too large to rotate fast enough to relieve the build-up of torsional stress. Cells use protein DNA topoisomerases to relieve the tension by producing transient nicks in the DNA backbone. The sliding clamp keeps DNA polymerase firmly attached to the template while is synthesising new strands of DNA. This protein forms a clamp around the “new” double helix and allows the enzyme to move along the template. On the other hand, the clamp loader hydrolyses ATP each time it locks a sliding clamp around a newly formed DNA double helix.

DNA REPAIR

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Even with proof-reading, DNA polymerases make too many errors (about 100-1000 per cell division in humans). Many mutagens (chemicals, UV light, etc…) continuously damage DNA, generating thousands of lesions per cell per day. Genetic alterations can be detrimental.

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Through the work of protein machines that continually scan the genome for damage, individuals remain, generally, genetically stables. Most DNA damage is only temporary and is corrected by DNA repair.

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DNA is continually undergoing thermal collisions with other molecules, resulting in chemical changes. Depurination and deamination are the most frequent chemical reactions known to create serious DNA damage in cells. Depurination can remove guanine or adenine from DNA. The major type of deamination reaction converts cytosine to an altered DNA base, uracil; however, deamination can also occur on other bases as well. Both depurination and deamination take place on the double-helical DNA, and neither break the phosphodiester backbone.

Ultraviolet radiation in sunlight promotes covalent linkage between two adjacent pyrimidine bases, forming a thymine dimer. The failure to repair this causes the disease xeroderma pigmentosum. This is a widely studied disease arising from defects in nucleotide excision repair. Patients are very sensitive to light, and UV-induced DNA damage. Skin cancers are common. 

Chemical changes, if left unrepaired, would lead either to the substitution of one nucleotide pair for another or to a deletion of one or more nucleotide pairs in the daughter DNA strand after DNA replication. DNA can be altered by replication itself by failing proofreading.

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The mechanisms of repair rely on the action of enzymes and on the double-helical structure of DNA. If the sequence in one strand is damaged, information is not lost because a backup version is conserved in the complementary nucleotide sequence in the other strand. Pathways for repairing damaged DNA: Double strand break repair  Base-excision repair  Nucleotide-excision repair.

Causes of DNA breaks: Exogenous - Radiation; - Chemicals. Endogenous - Oxygen: free radicals; - DNA Replication. Specialized - V(D)J recombination; - Class switching; - Meiosis. Outcomes after DNA break formation involve cell death, genome instability resulting in carcinogenesis and/or successful repair.

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During replication, DNA accumulates errors. Adult humans contain around 1014 cells. The human (diploid) genome = c. 6 x 109 base pairs. DNA replication error rate = c. 1 in 10 10. So: every two cell divisions there will, on average, be ≥1 error in DNA replication, generating an adult human requires about 45-50 cell divisions (2 46 = 7 x 1013), starting from the fertilised egg. So: on average an adult human cell contains c. 22-25 new mutations acquired since fertilisation.

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•

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+ If 95% of DNA is “junk”, most

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There will actually be far more than

mutations won’t be in coding

45-50 cell divisions over a lifetime,

regions (but 1-2 still will be; and

because

some “junk” matters);

continuously (skin);

Even in coding regions, many

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some

cells

divide

Mutations don’t just arise from

mutations don’t change protein

DNA replication errors (chemicals,

function;

UV, etc…).

We have two copies of each gene....