DNA and Replication Lecture 24 (Queen Mary, 2024) PDF

Fundamentals of Medicine DNA Structure and Replication MBBS, year 1 K. Kulesza-Smith ([email protected]) 30th September 2024  Learning Objectives Describe, using simple diagrams, the structure of DNA and its organisation into nucleosomes, chromatin and chromosomes. Outline...

Fundamentals of Medicine DNA Structure and Replication MBBS, year 1 K. Kulesza-Smith ([email protected]) 30th September 2024  Learning Objectives Describe, using simple diagrams, the structure of DNA and its organisation into nucleosomes, chromatin and chromosomes. Outline the mechanism of DNA synthesis (replication) and describe how some antibiotics interfere in this process. Explain the very low level of mistakes in the DNA replication process. Outline methods of DNA repair, with examples of clinical consequences of defective repair mechanisms. Reading: Molecular Biology of the Cell, 6th edition Bruce Alberts et al., New York: Garland Science; 2014. 2 Why DNA? Function: genetic material stores information Molecular basis for inheritance Contains the code for synthesis and regulation all Genes - blueprint for building other cellular molecules proteins Its complementary structure allows it to be DNA DNA RNA  proteins replicated and the code to be read Variations in DNA sequence lead to phenotypic (observable characteristics or traits) differences and susceptibility to disease Defects in DNA replication and repair lead to many diseases proteins 3  DNA Deoxyribo Nucleic Acid One molecule of DNA, consists of 2 strands of repeating units called nucleotides The 2 strands are twisted into a double helix 4 Nucleotides Each nucleotide contains: a deoxyribose (a pentose = five-carbon sugar) a nitrogenous (nitrogen-containing) base a phosphate group nucleotide nucleoside 6  Polynucleotides Condensation reaction! 7 Nitrogenous bases Derived from Purine Adenine Guanine nine-membered, double-ringed structures Derived from Pyrimidine Cytosine Thymine only found in DNA Uracil only found in RNA six-membered, single-ringed structures 8  Nitrogenous bases Purines y Pyrimidines DNA molecules exist as a double helix 2 antiparallel polynucleotide strands held together by hydrogen bonds between complementary base pairs The four DNA bases A C T and G form y base pairs such that; A can pair with T A=T C can pair with G C≡G i.e a purine with a pyrimidine. Purine = Huge Pyrimidines = Tiny y 9 The structure of DNA: pyrimidines- single ring 10 The structure of DNA: purines – double ring 11 DNA structure DNA has three components - - phosphate group pentose sugar (deoxyribose) nitrogenous base 12  The structure of DNA: polynucleotides Consists of covalently linked deoxyribonucleotides Linked by phosphodiester bridges between the 5’-hydroxyl group of one nucleotide and the 3’-hydroxyl group of the next Is the basis of the template (genome) from which all proteins are synthesised 13  And so we have our DNA model… The DNA helix is anti- parallel Each strand runs in opposite directions; -one runs 5’ (5 prime) to 3’ (3 prime) -the other runs 3’ to 5’ 14 Too much DNA? The nuclei of eukaryotic cells contain several linear chromosomes. The challenge: The diameter of the nucleus of a human cell is about 15 μm The total length of the naked DNA in this nucleus is about 7.5 feet or 2.3m. How does the cell make this fit? 15 Compaction of DNA 16  Short region DNA double helix Compaction Nucleosomal fibre- ‘beads on a of DNA string’ Chain of nucleosomes is then wrapped into a 30 nm spiral Histone H1 called a solenoid, where linker additional H1 histone proteins are associated with each nucleosome to maintain the chromosome structure. X1000 ~10 000x 46 metaphase chromosmes 17 DNA replication The enzyme gyrase (a topoisomerase enzyme) unwinds the DNA helix The enzyme helicase breaks the hydrogen bonds holding the base pairs together. 18 Free Nucleotides Bind Complementary base pairing DNA helicase completes the splitting of the strand. Meanwhile, free nucleotides that have been activated are attracted to their complementary bases. Each chain acts as a template. 19 DNA polymerase ’ A primer enzyme (Primase) binds a the start of the sequence to indicate where DNA polymerase should bind ’ Once in placed the activated nucleotides are joined together by DNA polymerase. ’ DNA polymerase joins the new nucleotides to each other by strong covalent bonds, forming the phosphate-sugar backbone. 20  Replication complete ’ The result is that there are two DNA molecules, each with one new synthesised strand of DNA and one strand from the original. ’ The DNA is then rewound by another enzyme. 21 Let’s look closer at the bigger picture… DNA polymerase works in a 5’-3’ direction This is an issue as one strand is the ‘wrong way round’! 22 Leading and lagging 23 24 Image result for dna replication 25 In eukaryotic cells there are hundreds to thousands of origins of replication- where Bacterial genome has a single replication is initiated. origin of replication. 26 Interference of DNA relocation by some antibiotics ORIGIN ↓ ’ 5 3’ target bacteria's 3’ 5’ single spots of replication New nucleotides added Topoisomerase prevents supercoiling of DNA 27 Quinolones target bacterial type II topoisomerases Unwound region DNA replication Quinolones (urinary and respiratory tract) Topoisomerase prevents supercoiling of DNA Overwound region (controls under and over Quinolones – leads to winding). Removes knots supercoiling of DNA and and tangles in bacterial double strand breaks in chromosome. Gram negative bacteria Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. Nat Rev Microbiol. 2010 Jun;8(6):423-35. doi: 10.1038/nrmicro2333. Epub 2010 May 4 28 DNA polymerase has separate sites for DNA synthesis and editing POLYMERISATION PROOFREADING OR EDITING 29 Proofreading and Editing DNA. DNA polymerase 1- error rate is DNA polymerase 1 in 100 000 bases 5’ 3’ New DNA strand 3’ 5’Template DNA strand Proofreads and corrects mistakes Repairs mismatched bases POLYMERASE ADDS INCORRECT NUCLEOTIDE Removes abnormal bases Error rate reduced to: 1 in 100 million bases MISREPAIRED NUCLEOTIDE REMOVED BY POLYMERASE (3’ TO 5’ EXONUCLEASE ACTIVITY) CORRECTLY PAIRED 3’ END ALLOWS ADDITION OF NEXT NUCLEOTIDE SYNTHESIS CONTINUES IN 5’ TO 3’ DIRECTION 30 Methods of DNA repair Single strand repairs Double strand repairs DNA Repair 31 Types of DNA repair Mechanisms Single strand defects – if not repaired lead to mutations Double strand breaks – if not repaired leads to genetic instability 32 Base Excision Repair Correction of small lesions (single bases) that do not significantly distort the DNA helix structure deaminated C 5’ G CTUATC C 3’ NH2 3’ C G AG TAG G 5’ URACIL DNA GLYCOSYLASE – “pinch, push and pull” G CT ATC C C G AG TAG G SUGAR PHOSPHATE REMOVED G CT ATC C C G AG TAG G DNA POLYMERASE ADDS NEW NUCLEOTIDES, DNA LIGASE SEALS THE NICK G CTC ATC C C G AG TAG G 33 Nucleotide Excision Repair pyrimidine dimer V 5’ C T A C G G T C T A C T A T G G 3’ 3’ G A T G C C A G A T G A T A C C 5’ NUCLEASE V C TAC G G TC TA C TATG G GAT G C CAGAT G A TAC C DNA HELICASE V C G G T C TA C TATG G C TAG 12 NUCLEOTIDE GAP G GAT G C CAGAT G A TAC C DNA PLOYMERASE AND DNA LIGASE 5’ C T A C G G T C T A C T A T G G 3’ 3’ G A T G C C A G A T G A T A C C 5’ 34 DNA mismatch repair DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base- base mismatches and insertion/deletion mis pairs generated during DNA replication and recombination. 35 DNA mismatch repair Newly made Nick = single strand break which Nick provide signals that directs the lagging strand Error in newly made strand mismatch proof reading system to the correct strand. BINDING OF MISMATCH PROOFREADING PROTEINS I MutS detects MutS binds to mismatched base pair DNA SCANNING DETECTS NICK MutL scans nearby DNA for scans ( MutL IN NEW DNA STRAND nick and triggers strand removal to the mismatch Individuals who inherit one STRAND REMOVAL defective copy of a mismatch repair gene have a predisposition for specific types of cancer. DNA REPAIR SYNTHESIS 36  Especially dangerous as there is no intact Double strand breaks template strand Without repair there would be rapid breakdown of chromosomes and loss of genes during cell division Cancer cells Normal cells Non-homologous Homologous end joining recombination 37 Double strand break repair by end-joining ACCIDENTAL BREAK LOSS OF NUCLEOTIDES DUE TO DEGRADATION FROM THE END (By the age of 70 – typically >2000 ‘scars’ throughout genome) SISTER CHROMOSOME COPYING PROCESS INVOLVING HOMOLOGOUS RECOMBINATION REGION WITH ALTERED SEGMENT DUE TO MISSING NUCLEOTIDES Homologous recombination COMPLETE SEQUENCE RESTORED BY COPYING FROM SISTER CHROMOSOME more accurate 38 Examples of inherited DNA repair defects Name Phenotype Affected processes Xeroderma Skin cancer, cellular Nucleotide excision pigmentosum (XP) UV sensitivity, repair neurological abnormalities MutS, MutL Colon cancer Mismatch repair BRCA2 Breast and ovarian Repair by cancer homologous recombination 39  Learning Objectives Describe, using simple diagrams, the structure of DNA and its organisation into nucleosomes, chromatin and chromosomes. Outline the mechanism of DNA synthesis (replication) and describe how some antibiotics interfere in this process. Explain the very low level of mistakes in the DNA replication process. Outline methods of DNA repair, with examples of clinical consequences of defective repair mechanisms. Reading: Molecular Biology of the Cell, 6th edition Bruce Alberts et al., New York: Garland Science; 2014. 40 Fundamentals of Medicine DNA Structure and Replication MBBS, year 1 K. Kulesza-Smith ([email protected]) 30th September 2024