MOL 4_HF_DNA replication repair_2324.pptx

Lecture 4 DNA Replication and Repair MOL 114 Lippincott’s Illustrated Reviews: Cell and Molecular Biology, Ch. 7 Dr Hanaa Fakhoury Hajeer  Main objectives Explain the process of DNA replication Identify the causes of DNA damage and explain repair mechani...

Lecture 4 DNA Replication and Repair MOL 114 Lippincott’s Illustrated Reviews: Cell and Molecular Biology, Ch. 7 Dr Hanaa Fakhoury Hajeer  Main objectives Explain the process of DNA replication Identify the causes of DNA damage and explain repair mechanisms Describe the effects of failure in DNA repair Link these concepts to the pharmacology of antivirals and cancer treatments Questions to be answered at the end of the lecture How certain antibiotics, antiviral and anti-cancer treatment can target DNA replication?  Primary structure of DNA DNA is a polymer of nucleotides linked by 3'→5'phosphodiester bond EUKARYOTIC DNA REPLICATION When cells divide, each daughter cell must receive an accurate copy of the genetic information DNA replication is the process in which each chromosome is duplicated before cell division STEPS IN PROKARYOTIC DNA SYNTHESIS Semiconservative replication Each of the individual parental strands remains intact in one of the two new duplexes DNA replication animation  Origins of replication Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” A eukaryotic chromosome may have hundreds or thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied Origin of replication  Formation of the replication fork Proteins required for DNA strand separation: – DNA helicase – Single- stranded DNA- binding protein Supercoiling resulting from DNA strand separation Unwinding at the replication fork causes supercoiling As helicase unwinds the DNA at replication forks, DNA ahead of it becomes overwound and supercoils form  Action of DNA topoisomerases They have strand- cutting and strand- resealing activities Type I DNA topoisomerases Type II DNA topoisomerases (DNA gyrase in prokaryotes) Topoisomerase inhibitors Inhibitors of prokaryotic topoisomerase II: Fluoroquinolones (nalidixic acid, ciprofloxacin, levofloxacin)are used as antibiotics  Inhibitors of eukaryotic topoisomerase II are used as anti-cancer treatment  etoposide and teniposide  Daunorubicin and doxorubicin 13  DNA Polymerase Enzyme responsible for copying the DNA templates Can only “read” the parental nucleotide sequences in the 3'→5' direction Can synthesize the new DNA strands only in the 5'→3' direction DNA polymerases catalyze the elongation of new DNA at a replication fork  Incorporation of a nucleotide into a DNA strand New strand Template strand 5end 3end 5end 3end Sugar A T A T Phosphate Base C G C G G C G C DNA polymerase 3end A T A T C Pyrophosphate 3end C Nucleoside triphosphate 5end 5end DNA polymerase proofreading DNA polymerases can correct mistakes ("proofreading") DNA polymerases have 3'→ 5' exonuclease activity for proofreading  RNA primer and primase DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. They require an RNA primer, which is a short, double-stranded region consisting of RNA base-paired to the DNA template The primer is short (5–10 nucleotides long), and the 3end serves as the starting point for the new DNA strand RNA primer is synthesized using primase enzyme  Leading and lagging strand Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase  Overview Lagging Origin of replication Leading strand Lagging strand strand 2 1 Leading strand Overall directions of replication Lagging strand synthesis  Telomeres Telomeres are non-coding and repetitive sequences at the ends of linear DNA molecules in eukaryotic chromosomes With each round of replication in most normal cells, the telomeres are shortened because DNA polymerase cannot complete synthesis of the 5' end of each strand This contributes to the aging of cells, because eventually the telomeres become so short that the chromosomes cannot function properly and the cells die  5 Ends of parental Leading strand DNA strands Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Parental strand Removal of primers and replacement with DNA where a 3end is available 5 3 Second round of replication 5 New leading 3 strand New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules  Telomere length and health There is a correlation between telomere shortening and human aging, disease progression, and developmental delay. Decreased telomere length in peripheral blood leukocytes has been shown to correlate with higher mortality rates in older (>60 years of age) individuals. There is also a correlation between telomere length and psychological stress and the risk for development of psychiatric disease.  Telomerase Telomerase is an enzyme in eukaryotes used to maintain the telomeres Telomerase replace telomere sequences that would otherwise be lost during replication A lack of telomerase activity in mammalian somatic cells results in progressive shortening of telomeres with each cellular replication event. Telomerase activity is present only in embryonic cells, germ (reproductive)cells, and stem cells, but not in normal somatic cells Cancer cells often have relatively high levels of telomerase, preventing the telomeres from becoming shortened and contributing to the immortality of malignant cells  Nucleoside analogs DNA chain growth can be blocked by the incorporation of certain nucleoside analogs that have been modified in the sugar portion of the nucleoside By blocking DNA replication, these compounds slow the division of rapidly growing cells ( cancer) and viruses ( HIV, Herpes) 24 Nucleoside analogs (HIV treatment) Substitution of the hydroxyl group of the deoxyribose with N3 as seen in AZidoThymidine (AZT), also called zidovudine Removal of the hydroxyl group from the 3′-carbon of the deoxyribose ring as in 2′,3′-dideoxyinosine 25 ([ddI] also known as  More nucleoside analogs Adenine arabinoside (or araA, vidarabine) is an antiviral agent (against herpes) Cytosine arabinoside (or araC, cytarabine) has been used in anticancer chemotherapy 26  DNA repair Multiple repair systems have evolved, allowing cells to maintain the sequence stability of their genomes Any defect in DNA repair carries an increased risk of cancer 27  Nucleotide excision repair Ultraviolet light induces the formation of dimers between adjacent thymines in DNA (also occasionally between other adjacent pyrimidines) The formation of thymine dimers interferes with DNA replication and normal gene expression Thymine dimers are eliminated from DNA by a nucleotide excision-repair mechanism 28 Thymine dimers 29  Pathology: Xeroderma pigmentosum Xeroderma pigmentosum is a genetic disease Caused by mutation in the nucleotide repair enzymes Defective repair of thymidine dimers It is an autosomal recessive disorder (incidence 1/250,000) characterized by extreme sensitivity to sunlight, skin freckling, ulcerations, and skin cancer Carcinomas and melanomas appear early in life, and most patients die of cancer Patients with the disease should avoid exposure to any source of UV light 30 Piccione, Monica, Anna Belloni Fortina, Giulia Ferri, Gloria Andolina, Lorenzo Beretta, Andrea Cividini, Emanuele De Marni, Francesca Caroppo, Ugo Citernesi, and Rosa Di Liddo. 2021. "Xeroderma Pigmentosum: General Aspects and Management" Journal of Personalized Medicine 11, no. 11: 1146. https://doi.org/10.3390/jpm11111146  Mismatch Repair Sometimes replication errors escape the proofreading function during DNA synthesis, causing a mismatch of one to several bases When the strand containing the mismatch is identified, the mismatched nucleotide is removed by an exonuclease 32  Nonpolyposis colorectal cancer Mutation to the proteins involved in mismatch repair in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome Pastor, Danielle M., and Jeffrey Schlom. "Immunology of lynch syndrome." Current Oncology Reports 23 (2021): 1-20. 33  Useful links https://ocw.mit.edu/courses/biology/7-0 1sc-fundamentals-of-biology-fall-2011/ molecular-biology/dna-replication/ http://www2.le.ac.uk/departments/gene tics/vgec/schoolscolleges/lectures/replic ation-recombination-and-repair-1