Lecture 2 - Week 2: Genome Organisation & Replication PDF

Genome organisation, structure & replication BI1CMP1 Dr Susanna Cogo [email protected] Lecture content: from DNA to proteins Genetic material must:​ contain complex information,​...

Genome organisation, structure & replication BI1CMP1 Dr Susanna Cogo [email protected] Lecture content: from DNA to proteins Genetic material must:​ contain complex information,​ replicate (copy itself) faithfully,​ encode all phenotypes of the organism,​ and have the capacity to vary. Central dogma – Information cannot be transferred from protein to protein or protein to nucleic acid but can be transferred between nucleic acids and from nucleic acid to protein.​ The translation of RNA into protein is unidirectional.​ Overview Lecture 2 – Lecture 3 – Lecture 4 – Week 2: Week 3: Week 4: Genome Gene The genetic organisation, expression, code, structure and transcription transcription replication Focus on eukaryotic cells Intended Learning Outcomes (ILOs) At the end of this lecture, you will be able to: Describe the structure of nucleic acids Compare and contrast the main functions and features of DNA and RNA Explain the double-helix structure of DNA Outline the main steps of eukaryotic DNA replication Discuss the importance of accurate DNA replication Activity Intended Learning Outcomes (ILOs) At the end of this lecture, you will be able to: Describe the structure of nucleic acids Compare and contrast the main functions and features of DNA and RNA Explain the double-helix structure of DNA Outline the main steps of eukaryotic DNA replication Discuss the importance of accurate DNA replication Further reading/study material Let’s get started True or false Key definitions A genome is a complete set of genetic instructions for any organism – either RNA or DNA​. Copied during process of replication.​ The coding system for genetic information is the same in all living organisms. RNA is the bridge between genes and the proteins for which they code​. Transcription is the synthesis of RNA using information in DNA​. Transcription produces RNA (mRNA for protein coding genes)​. Translation is the synthesis of a polypeptide, using information in the mRNA.​ Ribosomes are the sites of translation. The route to the discovery of DNA and its structure The route to the discovery of DNA and its structure By the end of 19th century researchers concluded form the observations of Gregor Mendel and others that the genetic material is contained in cells​. 1869: Friedrich Miescher isolated a substance from cell nuclei, that he called nuclein.​ 1928: Frederick Griffith demonstrated the transmission of genetic instructions by a process called the "transformation principle".​ 1944: the team of Avery, MacLeod and McCarty suggested that DNA is the “transforming factor” and not proteins or other materials.​ 1952: Hershey and Chase proved that DNA was the genetic material and not protein in bacteriophage. https://www.youtube.com/watch?v=92HWRdvDF_s James Watson & Francis Crick 1951: Maurice Wilkins and Rosalind Franklin produced a picture of the DNA molecule using X-ray crystallography ​ James Watson and Francis Crick – Molecular structure of nucleic acids (Nature, 1953) In 1962 Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine for their determination of the structure of DNA. Different living organisms, common themes Physical separation between organism and environment, which defines self and non-self. Stable but flexible storage of the genome. Reliable replication and transmission of information, required for the generation of both offspring and tissues. A source of energy from their surroundings to grow and reproduce (e.g. sun for plants, food for humans). Different living organisms, common themes Physical separation between organism and environment, which defines self and non-self. Stable but flexible storage of the genome. Reliable replication and transmission of information, required for the generation of both offspring and tissues. A source of energy from their surroundings to grow and reproduce (e.g. sun for plants, food for humans). DNA vs RNA Nucleic acids are essential macromolecules in the continuity of life: DNA (deoxyribonucleic acid) carries the genetic inheritance of a cell, and instructions for the functioning of the cell, while RNA (ribonucleic acid) communicates this information to the rest of the cell. RNA DNA & RNA are polymers composed of monomers termed the nucleotides (nitrogen base + sugar phosphate backbone). The nucleotides composing DNA are deoxyguanosine monophosphate (G), deoxyadenosine monophosphate (A), deoxythymidine monophosphate (T), and deoxycytidine monophosphate (C). Nucleotide structure Ionized hydroxyl groups Phosphate group(s) Five-carbon sugar Nitrogenous base Nucleotides are organised in strands Polynucleotide chains have nitrogenous bases linked to a sugar-phosphate backbone Nucleotides are linked by phosphodiester bonds (C-O-P-O) to form a DNA strand Phosphodiester bonds of the DNA give the polarity of the DNA strand (5’ phosphate and 3’ hydroxyl end) Building DNA molecules The sequence of the 4 nucleotides contains the genetic information, in a structure termed the (antiparallel) double-helix. A & G = purine (2 rings) DNA atomic models (adapted from Marini C & T = pyrimidine (1 ring) et al., Science Advances, 2015) The structure of DNA is a double-helix Hydrogen bonds between the bases of the opposite strands and base stacking of bases within a strand contribute to the stability of DNA double helix The structure of DNA is a double-helix The B-form of DNA is a double helix consisting of two polynucleotide chains that run antiparallel. The diameter of the double helix is 20 Å. There is a complete turn every 34 Å, with 10 base pairs per turn. The double helix has a major (wide) groove and a minor (narrow) groove. The two strands of DNA are © Photodisc complementary: base pairs that match up in the pairing reactions in double helical nucleic acids (A with T, and C with G). What does an Å correspond to? The structure of DNA is a double-helix The B-form of DNA is a double helix consisting of two polynucleotide chains that run antiparallel. The diameter of the double helix is 20 Å. There is a complete turn every 34 Å, with 10 base pairs per turn. The double helix has a major (wide) groove and a minor (narrow) groove. The two strands of DNA are © Photodisc complementary: base pairs that match up in the pairing reactions in double helical nucleic acids (A with T, and C with G). 1Å = 1×10−10 m = 0.1nm The base pairing is specific Watson and Crick reasoned that the pairing was specific, dictated by the base structures. Purine + purine: too wide A & G = purine (2 rings) C & T = pyrimidine (1 ring) Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Conclusion: adenine (A) paired only with thymine (T), guanine (G) paired only with cytosine (C) Chargaff’s rules (1951): in any organism the amount of A = T, and the amount of G = C; and the number of purines (A+G) = the number of pyrimidines (C+T) Analogies and differences between DNA and RNA Analogies and differences between DNA and RNA deoxyribonucleic acid/ DNA ribonucleic acid/ RNA DNA replicates and stores RNA replicates and stores genetic genetic information information, and converts the DNA consists of two strands, genetic information contained within DNA to a format used to arranged in a double helix, made build proteins up of nucleotide subunits RNA only has one strand, but like DNA is a much longer polymer DNA, is made up of nucleotides than RNA (a chromosome is a single, long DNA molecule which RNA molecules are variable in could be centimetres in length) length, but much shorter than The sugar in DNA is deoxyribose, long DNA polymers which contains one less hydroxyl RNA contains ribose sugar group than RNA’s ribose molecules The bases in DNA are Adenine RNA shares Adenine (‘A’), (‘A’), Thymine (‘T’), Guanine (‘G’) Guanine (‘G’) and Cytosine (‘C’) and Cytosine (‘C’) with DNA, but contains Uracil (‘U’) rather than Thymine Chromosomes and chromatin If stretched out, DNA would be 2m long. Hence, it needs to be reorganised in a compact structure able to fit in the cell nucleus. DNA is packed with proteins (histones) in a highly compact shape: chromatin. Histones package the massive amount of DNA in a genome into a highly compact form. Nucleosomes are complexes of DNA and 8 histones. https://www.youtube.com/watch? v=6Z8aQhV_aD4&t=50s Open vs closed chromatin Chromatin exists in an open (euchromatin) and a closed (heterochromatin) conformation Chromatin remodelling allows DNA to be exposed in the open conformation and accessed for replication, transcription, repair, etc. Chromatin remodelling is affected by many factors, including  Histone variants  Histone post-translational modification  ATP-dependent chromatin remodelling complexes. Learn more on histone post-translational modifications https://www.youtube.com/watch?v=XQ3P7Bq9ld0 Organelle DNA Some organelles have their own genome – and multiple copies of it!!! (e.g. mitochondria). 16569 bp and 37 genes. When and how is the information transmitted? The sequence of nucleotides in DNA defines the genetic information of an organism. The genomes of multiple organisms (both unicellular and multicellular) have been completely sequenced (the first one was Haemophilus influenzae in 1995). Before cell division, the entire DNA content must be copied, and then separated through segregation. The process needs to be accurate and fast. (What are the consequences of errors in the replication?) Coordinating DNA replication with the cell cycle DNA replication (a) Parental (b) Separation of parental (c) Formation of new strands molecule strands into templates complementary to template strands DNA replication is a semiconservative process Meselson and Stahl – DNA replication is semiconservative Semiconservative replication – DNA replication accomplished by separation of the strands of a parental duplex, each strand then acting as a template for synthesis of a complementary strand. The sequences of the daughter strands are determined by complementary base pairing with the separated parental strands. How does eukaryotic replication start? DNA has to be accessible Replication begins at origins of replication Many origins of replication Bidirectional - replications fork move towards each other 500-5000 nucleotides/minutes (bacteria are 10 times faster!!!) Linear DNA replication Replication progresses in the direction 5’ to 3’ Initiator proteins separate DNA strands for access of the DNA helicase DNA helicase “opens” the DNA helix, breaking hydrogen bonds Topoisomerase unwinds the DNA molecule Primase synthetises short (about 10nt) RNA stretches (primers) which provide a 3’-OH group to start the replication Elongation requires free dNTPs (deoxynucleoside triphosphates) and the action of DNA polymerases The addition of dNTPs to a new DNA molecule Each nucleotide that is added to a growing DNA strand is part of a dNTP molecule. dNTP has deoxyribose while NTP has ribose As each monomer of dNTP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate  DNA synthesis is continuous on one strand (the leading strand), discontinuous on the other (the lagging strand) In the lagging strand, shorter fragments are synthetised (100-200 nucleotides in eukaryotic genes) and more primers are required Final steps The combined action of exonuclease and DNA polymerase catalyses the replacement of RNA primers with DNA dNTPs DNA ligase joins the ends of Okazaki fragments What guarantees the accuracy of the process? DNA polymerases also possess a) proofreading and b) exonuclease activities, to limit errors (only 1 in 10 million nucleotides is incorrectly added). Further repair mechanisms are happening once the replication is completed. Uncorrected errors can lead to mutations. Replicating the ends of linear DNA molecules Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes The usual replication machinery provides no way to complete the 5′ ends, so repeated rounds of replication produce shorter DNA molecules with uneven ends This is not a problem for prokaryotes, most of which have circular chromosomes Telomeres Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends called telomeres Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules It has been proposed that the shortening of telomeres is connected to aging DNA repair mechanisms (in a nutshell) A mutation is an inherited alteration in the DNA sequence (can be inherited within the same organism and through generations) Multiple factors can cause mutations, including errors in replication or the action of chemicals which damage DNA DNA repair mechanisms are in place to correct mutations More on DNA repair https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474181/ Learning from nature – the development of DNA technologies 1983: Kary Mullis develops the polymerase chain reaction (PCR). Awarded the Nobel Prize in 1993 together with Michael Smith (site-directed mutagenesis). https://www.youtube.com/watch?v=c07_5BfIDTw https://www.youtube.com/watch?v=vBDH6L2vmok 2011: CRISPR-Cas9 genome editing. Nobel Prize awarded in 2020 to Emmanuel Charpentier and Jennifer Doudna. https://www.youtube.com/watch?v=UKbrwPL3wXE https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070239/ True or false Further reading https://www.nature.com/scitable/topicpage/discovery-of-dna-structure-and-function-watson-397/ Additional books available as hard copy or online resource from the library!