Biol1110 2022 Lecture 4 - DNA Replication PDF

Biol1110 Genes to Organisms DNA replication “So that's when I saw the DNA model for the first time, in the Cavendish, and that's when I saw that this was it. And in a flash you just knew that this was very fundamental. - Sydney Brenner Lec...

Biol1110 Genes to Organisms DNA replication “So that's when I saw the DNA model for the first time, in the Cavendish, and that's when I saw that this was it. And in a flash you just knew that this was very fundamental. - Sydney Brenner Lecture 4 – DNA replication  Lecture outline DNA replication Telomeres DNA mutation, damage and repair Lecture 4 – DNA replication  Divide and conquer Prokaryotes (and some eukaryotes) are single cells – Cell division = reproduction Most eukaryotes are multicellular – But we start from a single fertilized egg – Cell division is necessary for growth and development – One cell à approximately 100,000 billion cells in an adult human (1013) – Also necessary for repair Cell division requires DNA replication Lecture 4 – DNA replication Copying mechanism “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” - Watson and Crick Nature, 171, 737-8 Lecture 4 – DNA replication Copying mechanism ATGCGCCGTA TACGCGGCAT Lecture 4 – DNA replication  Copying mechanism ATGCGCCGTA TACGCGGCAT ATGCGCCGTA TACGCGGCAT Lecture 4 – DNA replication  Copying mechanism ATGCGCCGTA TACGCGGCAT ATGCGCCGTA TACGCGGCAT ATGCGCCGTA ATGCGCCGTA TACGCGGCAT TACGCGGCAT Lecture 4 – DNA replication  Copying mechanism ATGCGCCGTA TACGCGGCAT ATGCGCCGTA How? TACGCGGCAT ATGCGCCGTA ATGCGCCGTA TACGCGGCAT TACGCGGCAT Lecture 4 – DNA replication  DNA replication Base pairing is all important –C–G –A–T But most chromatin is usually tightly wound around histones – Need to unwind for replication Lecture 4 – DNA replication  Semi-conservative replication Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules semi-conservative model Lecture 4 – DNA replication  Replication via base-pairing Lecture 4 – DNA replication  Proteins involved in DNA replication Topoisomerase – breaks, rejoins, swivels the DNA Helicase – untwist and separates the DNA helix Primase – synthesises RNA primers DNA polymerase(s) – synthesises the new DNA strand Single Strand Binding (SSB) proteins – bind to single DNA strands and keep them from re-joining DNA ligase – joins the pieces of DNA together (forms phosphodiester bonds) Lecture 4 – DNA replication Lecture 4 – DNA replication  DNA 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 even thousands of origins of replication Replication proceeds in both directions from each origin Lecture 4 – DNA replication Origin of replication Lecture 4 – DNA replication  Stages of DNA replication Initiation – Specific DNA sequence at which the helix is unwound and open to enzymes (proteins) – Helicases unwind the DNA strand Elongation – DNA polymerases add nucleotides to the growing DNA strand (500 nucleotides per second in bacteria, 50 per second in humans) Termination – End-point of replication (where replication forks meet) Lecture 4 – DNA replication Replication of the leading strand Lecture 4 – DNA replication Replication of the lagging strand Lecture 4 – DNA replication  Some more key terms RNA primer – Short strand of RNA complementary to DNA & enables the binding of DNA polymerase, 5 – 10 nucleotides long RNA primase – Enzyme which synthesizes RNA primers Okazaki fragment – Short, newly synthesized DNA fragments that are formed on the lagging strand Lecture 4 – DNA replication  Some more key terms Leading strand – DNA which is being synthesized in the same direction as the growing replication fork Lagging strand – DNA whose direction of synthesis is away from the replication fork Ligase – Enzyme which joins together Okazaki fragments Lecture 4 – DNA replication  Antiparallel elongation The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication DNA polymerases add nucleotides only to the free 3¢ end of a growing strand; therefore, a new DNA strand can elongate only in the 5¢ to 3¢ direction Lecture 4 – DNA replication  Antiparallel elongation Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork Along the other template strand, a new lagging strand is synthesized non-continuously Lecture 4 – DNA replication  Overview of DNA replication Video Lecture 4 – DNA replication  Errors during DNA replication Errors occur at a rate of 1 in 105 nucleotides But, changes to the sequence (mutation) only occurs in around 1 in 1010 nucleotides This is due to proof-reading and repair mechanisms Errors that persist in DNA are called mutations Lecture 4 – DNA replication The telomere problem 26  TheTelomerase telomere action problem Telomerase carries a short RNA template and adds complementary DNA sequence to the 3’ ends of the chromosomes Telomeres contain no genes but many non-coding short repeats, in humans the repeat unit is 5’-TTAGGG-3’n 27  The telomere problem Not an issue for circular DNA Telomeres are repeated sequences at the end of chromosomes, e.g. TTAGGG repeated 100- 1000 times in humans Telomerase (= a protein) helps maintain the length of telomeres Lecture 4 – DNA replication  Mutations Mutations – Are changes in the genetic material of a cell – Are the source of new genes – Are the raw material of evolution and natural selection – May be positive (beneficial), negative (deleterious) or neutral Mutations can occur on a small scale – Affecting one or a few nucleotides Or on a large scale – Affecting large sections of chromosomes Lecture 4 – DNA replication  Mutations Mutations can occur from errors in DNA replication But also from mutagens – Physical agents X-ray UV rays – Chemical agents (carcinogens) Ethidium bromide Aflatoxin Tobacco smoke Asbestos Lecture 4 – DNA replication  Point mutations can affect protein structure and function Point mutations within a gene can be divided into two general categories – Base-pair substitutions – Base-pair insertions or deletions Lecture 4 – DNA replication  Point mutations can affect protein structure and function Lecture 4 – DNA replication Base-pair substitution: change of one base per DNA strand Silent mutation Causes no change in Insert Fig. 17-23 P347amino acid Missense mutation causes change in amino acid, loss or altered function of protein Nonsense mutation introduces stop codon Lecture 4 – DNA replication Base-pair insertion or deletion can cause a frame shift Frameshift causing nonsense introduces stop codon by insertion of a base Frameshift causing missense Series of wrong amino acids by deletion of a base No frameshift One codon (3 bases) are deleted or inserted Lecture 4 – DNA replication  Mutation and cell function 1. Loss-of-function mutation = mutation may produce proteins with impaired function that directly affects the cells that express that protein e.g. sickle cell anaemia caused by a single amino acid change in globin protein of haemoglobin results in the structural collapse of red blood cells normal red blood sickled red cells blood cells Lecture 4 – DNA replication  Mutation and cell function 2. Gain-of-function mutation = Produces a new trait or causes a trait to appear in inappropriate tissues or at inappropriate times in development. A 546 bp DNA sequence (gain-of-function mutation) in ancient humans led to the evolution of the opposable thumb. Science (2008). 321:1346 - 1350 Lecture 4 – DNA replication  Chromosomal mutations: alterations in chromosome structure Breakage of a chromosome can lead to four types of changes in chromosome structure: – Deletion removes a chromosomal segment – Duplication repeats a segment – Inversion reverses a segment within a chromosome – Translocation moves a segment from one chromosome to another Lecture 4 – DNA replication Summary chromosome rearrangements 38  DNA repair There are many DNA repair proteins – Over 100 in bacteria Mismatch repair proteins remove and replace incorrect nucleotides Nuclease – protein that cuts out the incorrect nucleotide Lecture 4 – DNA replication  Nucleotide excision repair Thymine dimers are caused by UV light Lecture 4 – DNA replication  Lecture summary DNA replicates in a semi-conservative manner There are numerous proteins involved in DNA replication (helicase, topoisomerase, primase, DNA polymerase, DNA ligase) DNA replication starts at the origin of replication DNA replication occurs along the leading strand and the lagging strand in anti-parallel elongation Lecture 4 – DNA replication  Lecture summary Shortening of DNA occurs in linear DNA, but telomeres and telomerase help maintain the length of DNA Errors in replication and mutagens can cause mutations; there are different types of mutations Mutations can be repaired using various mechanisms (nucleotide excision repair) Lecture 4 – DNA replication