DM Oral Biochemistry-SPBU- Lectures 9 & 10.pdf

1  Concept map of Function of nucleotides. Matrix biosynthesis Structure and function of Nucleic acid DNA Structure different types of structure Replication RNA...

1  Concept map of Function of nucleotides. Matrix biosynthesis Structure and function of Nucleic acid DNA Structure different types of structure Replication RNA transcription Regulation of gene Translation expression in Post prokaryotes and translational eukaryotes conversion  Intended learning Outcomes: Studying this chapter should enable you to: Describe the structure of a nucleotide Compare nucleotides with nucleosides Describe the flow of genetic information Describe the Physio-chemical properties of nucleic acids Realize that DNA replication is semiconservative, semicontinuous and bidirectional List the proteins involved in replication and outline their functions Name the components needed for DNA synthesis Label DNA replication fork drawing  Basic Structure of Nucleotide Nucleotide Pentose Nitrogenous Phosphate sugar base Nucleoside  Sugars RNA contains DNA contains ribose deoxyribose HO CH 2 OH HO CH 2 OH O O OH OH OH H D -ribose D-deoxyribose Found in RNA Found in DNA  Nitrogenous Bases Nitrogenous Bases Pyrimidines Purines Cytosine Uracil Thymine Adenine guanine  Cytosine DNA,RNA Pyrimidines Thymine DNA only Uracil RNA only Guanine DNA,RNA Purines Adenine DNA,RNA  Cleavage of phosphodiester bond The phosphodiester bonds can by enzymatically hydrolyzed by enzymes called nucleases Exonuclease Endonuclease Exinuclease  Concept map of Function of nucleotides. Matrix biosynthesis Structure and function of Nucleic acid DNA Structure different types of structure Replication RNA transcription Regulation of gene Translation expression in Post prokaryotes and translational eukaryotes conversion  DNA DNA structure Physio-chemical DNA function properties of DNA DNA structure Primary structure (polydeoxyribonucleotides) Secondary structure (DNA double helix) Tertiary structure (chromosomes)  Primary structure (Polydeoxyribonucleotides) Pentose sugar 2-deoxyribose. Nitrogenous bases A, G, C and T. Nucleotides d-AMP, d-GMP, d-CMP and d-TMP.  Secondary structure (DNA double helix) Features of the double helix: Double helix: The two strands are antiparallel: Base pairing rule: Spiral Staircase: Dimensions:  Double helix: The 2 strands of DNA are twisted around a common axis to form a right handed double helix. The two strands are antiparallel: One runs from 5`→ 3` direction and the other in 5’ 3’ the opposite direction from 3`→ 5`. T A G C A C A T C G T G 3’ 5’  Base pairing Rule and complementary bases A = T & C = G (Chargraff’s Rule)  The 2 strands are complementary.  so sequence of N2 bases in one strand determines the sequence of N2 bases in the other strand.  Base pairing Rule and complementary bases Adenine pairs with Guanine pairs with Thymine by 2 hydrogen Cytosine through 3 bonds hydrogen bonds Tertiary structure (chromosomes)  DNA package inside nucleus Huge Human genome (DNA is two meters long) Diameter of nucleus = 5-10 mm This long DNA interacts with many proteins (nucleoproteins) to be packed into the nucleus  From DNA (2ry structure) to Chromosomes (3ry structure) Chromosome Chromatin fibers nucleosomes DNA double helix  Functions of DNA DNA is able to replicate (DNA synthesis) during cell division. DNA has the specific genetic information to be selectively expressed by transcription (RNA synthesis) as the first step in gene expression.  Physio-chemical properties of Nucleic Acid DNA Denaturation Renaturation (Melting)  DNA Denaturation Separation of 2 DNA strands by disruption of hydrogen bonds between 2 DNA strands by: Change in PH Heating Denatured DNA ATGAGCTGTACGATCGTG ATGAGCTGTACGATCGTG ATGAGCTGTACGATCGTG TACTCGACATGCTAGCAC TACTCGACATGCTAGCAC Double stranded DNA Double stranded DNA TACTCGACATGCTAGCAC Single stranded DNA  Melting point (Tm) Point is reached at which 50% of DNA molecule exist as single strands G-C content of sample (high GC contents, high Tm )  Renaturation Complementary DNA strands separated by denaturation can reform double helix if cooled Central dogma of molecular biology  DNA Replication DNA Replication = DNA  DNA copying and transmission of the genetic information found in DNA to daughter cells.  DNA RNA according to instructions stored along a specific sequence (a gene) of a DNA molecule. The transcribed RNA will act as the working copy of the gene for protein synthesis. 一Translation or expression of the genetic information carried by the mRNA directs the ribosomes to synthesize a particular polypeptide (protein).  DNA unwound forming Replication overview 5’ replication fork Double-stranded DNA 3’ unwinds. base pair 5’ The junction of the a replication unwound molecules is fork. 3’ A new strand is formed complementary bases with by pairing the parent strand Two molecules are one new and one old DNA made, each has strand. Replication is forks move in both direction bidirectional away from the origin  DNA replication steps Separation of the 2 DNA strands (unwinding) Synthesis of new DNA strands  DNA replication requirements Let’s meet the team… DNA Free template nucleotides Enzymes ATP Replication steps: Separation of the 2 DNA strands Helicase (Unwind DNA) Cleaves the hydrogen bonds between the two DNA strands and requires energy provided by ATP. Untwisting and separation of the two strands of DNA Replication steps: Separation of the 2 DNA strands Single-strand binding proteins keep The 2 DNA strands apart (unpaired) They bind tightly to each of the separated strands, preventing them from rejoining Protect the template from nucleases that cleave single stranded DNA.  DNA supercoiling As the 2 strands are separated from each other, this creates coils in front of the separated part (supercoils) which prevents further separation of the helix.  DNA Toposiomerases Toposiomerases are enzymes that are responsible for the elimination of supercoils.  Clinical significance Quinolones antimicrobial drugs e.g nalidixic acid inhibit bacterial gyrase prevent bacterial replication and transcription  DNA replication steps Separation of the 2 DNA strands (unwinding) Synthesis of new DNA strands  Synthesis of new DNA strands DNA polymerases are a group of enzymes that are responsible for copying DNA templates by adding complementary deoxynucleotides to 3’OH of the growing chain. DNA Polymerases only synthesize DNA in one direction from 5’ to 3’ Players of New DNA strand synthesis  Okazaki Leading & Lagging strands Limits of DNA polymerase III  Unidirectional ,synthesizes DNA from 5 to 3 direction only 5′ 3′ 5′ 3′ 5′ 3′ 5′ 5′  3′ Lagging strand growing 3′ replication fork 5′ Leading strand 3′  5′ 3′ DNA polymerase III __________________  continuous synthesis  Leading strand synthesis continues in a 5’ to 3’ direction. 3’ Overall direction of replication 3’ 5’ 5’ 3’ 5’ 3’ 5’ Replication Overall direction 3’ of replication 3’ 5’ 5’ Okazaki fragment 3’ 3’ 5’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication Overall direction 3’ of replication 3’ 5’ 5’ Okazaki fragment 3’ 3’ 5’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication Overall direction 3’ of replication 3’ 5’ 5’ Okazaki fragment 3’ 3’ 5’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication Overall direction 3’ of replication 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Replication 3’ 3’ 5’ 5’ 3’ 5’ 3’5’ 3’5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.  End Result Formation of a complete, new daughter DNA strand that is complementary to the parent DNA strand  Proof Reading or Editing Proof Reading or Editing of the newly synthesized DNA (Replication fidelity): DNA polymerase, is self-correcting: it has a proofreading activity  To ensure Improper replication fidelity, DNA pol III and base pairing DNA pol I has in addition to 5’→3’ polymerase activity Dangerous 3’→5’exonuclease activity (proof mutations. reading activity)  DNA amplification can proceed either: In In vitro inside a vivo inside a test tube living cell Molecular e.g. PCR cloning 58  Polymerase Chain Reaction (PCR) In vitro method for amplification of a DNA segment segment (e.g. a gene) (e.g. in in a gene) a test tube a test to a tube million of copies imitating but(imitating in few hours DNA replication DNA in a test tube. replication but in a test tube) 59 Components in PCR reaction 1.Target DNA 2.Nucleotides (dNTPs) 3.DNA polymerase 4.Primers 60  PCR steps PCR occurs in repeated cycles, each cycle includes three steps: Denaturation. Annealing Extension. 95°C 55°C 70°C Annealing Separation of between Elongation of DNA double primer and DNA helix by heat DNA strands 61 Polymerase Chain Reaction(PCR) Number of DNA copies produced by PCR = 2Number of cycles Example: If number of cycle =4 Number of DNA copies=24= 16 DNA molecules 62  PCR cycles The cycle can be repeated 30 or more times. At the end of a cycle, each piece of DNA in the vial has been duplicated. Each newly synthesized DNA piece can act as a new template, so after 30 cycles, a million copies of a single piece of DNA can be produced! 63  PCR Applications Diagnosis of Forensic diseases by testing detection of pathogens 64  Activity 1 Answer the following questions guided by the provided figure of DNA structure. Q1- What is the type of the provided DNA structure? Q2- What is the name of the chemical bond (1)? Q3-Identify structure (2). 65  Activity 2 The provided figure is for PCR machine. Answer the following: Q1- List the components required for PCR. Q2-Give one example for DNA amplification in vivo. Q3-What is the importance of ( denaturation step ) in PCR? 66 67