DNA synthesis F23.pdf

DNA Synthesis – Ch 30 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Announcements • Exam Soft: • • Apple has announced a new version of their operating system, MacOS 14, Sonoma. Please note: Examplify is not compatible. To avoid issues with testing, stud...

DNA Synthesis – Ch 30 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Announcements • Exam Soft: • • Apple has announced a new version of their operating system, MacOS 14, Sonoma. Please note: Examplify is not compatible. To avoid issues with testing, students should not update their operating system until Examplify is compatible. Exam Challenge Representative: • • • One for Dent 604 and one for CMB 704 Collect all exam challenges and will provide justification for the question is being challenged. Send exam challenge document to Dr. Raucher 24hrs after the release of the exam and key. 2 Learning Objectives • Describe the composition and structure of a DNA molecule • Apply knowledge of nucleotide pairings to produce the complimentary sequence of a given nucleic acid sequence • Identify the enzymes involved in DNA replication and describe the activities of each of these enzymes • Explain the processes of leading strand synthesis and lagging strand synthesis • Explain the function of telomeres and telomerase • Describe the causes of DNA mutations and the major types of mutations • Compare and contrast the major types of DNA repair processes • Identify specific molecules involved with each type of DNA repair • Associate types of DNA mutations with the DNA repair process that cells would use to correct them 3 DNA Structure • DNA strands are composed of bases along a phosphate-sugar backbone • The backbones run in opposite directions - antiparallel • DNA is read along the backbone (5′ → 3′) • DNA is negatively charged • Complimentary bases held together via hydrogen bond 4 DNA Structure • Each nucleotide in the chain is connected via a phosphodiester bond • 3’-carbon of one sugar is connected to 5’-carbon of the next sugar via the phosphate group • Linkage yields free O- at physiological pH 5 DNA structure • During storage, DNA wraps around histone proteins — positively charged proteins (lysine, arginine) – Why? • This histone-wrapped DNA is stored in a highly condensed structure called chromatin • • Heterochromatin is tightly packed and inactive Euchromatin is unwound and active 6 Cell Cycle • DNA synthesis occurs during the S phase of interphase • Duplicate the chromosomes via DNA replication 7 DNA Replication is Semiconservative • DNA serves as the template for its own duplication 1. Two parent strands separate 2. Each parent strand serves as a template for the newly synthesized strand 3. Yields complete DNA molecule that is a hybrid of one ‘old’ strand and one ‘new’ strand 8 DNA Replication • Divided into 3 main steps 1. Initiation 2. Elongation 3. Termination 9 DNA Replication – Initiation • Occurs at specific nucleotide sequences – origins of replication (Ori) • • Ori’s rich in A-T base pairs – Why? DNA replication initiates at a single site in prokaryotes and at multiple sites in eukaryotes – Why? 10 DNA Replication – Initiation Proteins • • Group of proteins form prepriming complex Responsible for 1. Melting the bonds at the Ori 2. Maintain separation of parental strands 3. Unwind double helix Initiation Proteins in Prokaryotes 1. DnaA 2. DNA helicase (DnaB) 3. Single-stranded DNA-binding protein 4. Type I and II Topoisomerases 11 DNA Replication – Initiation Proteins 1. DnaA Protein • Binds DnaA Box (specific sequence) within Ori to initiate replication • Binding causes AT-rich region to melt • Melting and strand separation short region of ssDNA 12 DNA Replication – Initiation Proteins 2. DNA Helicase • Enzyme that binds to ssDNA at Ori • Moves toward double-stranded region to force the strands apart and unwinds double helix • Requires energy from ATP hydrolysis • Process results in supercoiling 13 DNA Replication – Initiation Proteins 3. Single-stranded Binding (SSB) Proteins • Binds ssDNA created by Helicase • Binding of first SSB protein facilitates binding of other SSB proteins – cooperative binding • Keep the strands separated in replication bubble • Protect DNA from nucleases that degrade ssDNA 14 DNA Replication – Initiation Proteins Topoisomerase 3. • Remove supercoils in DNA double helix by cleaving one or both DNA strands Topoisomerase I: A. Cuts one strand, rotates it around intact strand, and reseals the nick • Stores the energy from breaking a phosphodiester bond to reseal strand • Topoisomerase II: B. Binds tightly to DNA to make a transient break in both strands • Passes a different section of double helix through the break and reseals it • Requires ATP • DNA Gyrase in bacteria • 15 DNA Replication – Elongation • The site of replication called the replication fork • Both strands copied simultaneously • DNA pol III synthesize DNA only in the 5′ → 3′ direction • Leading strand synthesized continuously in the 5′ → 3′ direction • Lagging strand synthesized discontinuously (Okazaki fragments) in the 5′ → 3′ direction. 16 DNA Polymerase requires a Primer • Primer = short strand complementary to the template • contains a 3’-OH to begin the first DNA polymerase-catalyzed reaction • Primase synthesizes short RNA strand (~10 bp) complementary to DNA • DNA pol I removes primers and replaces RNA with DNA. 17 DNA Replication - Elongation • Leading strand • • continuous growth in 5’ to 3’ direction same direction as replication fork movement • Lagging strand • • • discontinuous growth in 5’ to 3’ direction copied in direction away from replication fork okazaki fragments are joined by ligase to form single strand 18 Trombone Model for Lagging Strand Synthesis • Lagging strand looped to pass through polymerase active site in the 3′ → 5′ direction • After ≈1000 nucleotides synthesized, loop released and new loop formed • Trombone Model • DNA polymerase I removes the RNA primer • DNA ligase joins the fragments to yield an intact strand. 19 DNA Replication - Elongation • DNA polymerase elongate DNA strand by adding dNTPs to 3’ end of growing chain • Sequence is determined by base sequence on parental (template) strand • All four dNTPs (A, T, G, C) must be present for elongation to occur 20 Proofreading New DNA Strand • DNA pol III has proofreading capability in 3’ to 5’ direction • • exonuclease activity 5’ to 3’ polymerase domain and 3’ to 5’ exonuclease domain are at different locations on DNA pol III 21 DNA Replication - Elongation 22 DNA Replication - Termination • Termination Utilization Substance (Tus) protein • • sequence-specific binding protein Tus binds replication Termination (Ter) sites on DNA to stop movement of replication fork • physical block 23 DNA Replication in Eukaryotes • ORC = origin recognition complex • MCM = minichromosome maintenance (complex) • RPA = replication protein A • PCNA = proliferating cell nuclear antigen • FEN = flap endonuclease. 24 Eukaryotic DNA Polymerases 25 Termination of Replication in Eukaryotes • • Telomeres • Found at linear ends of chromosomes (DNA) • Protect from damage by nucleases • Distinguishes end of chromosome from a double-stranded break • Shorten with each successive cell division • Somatic human cells divide about 52 times before losing the ability to divide again • Germ cells and stem cell telomeres do not shorten due to telomerase activity Telomerase (ribonucleoprotein): maintains telomeric length in cells • High telomerase activity is a characteristic of cancer cells 26 DNA Repair 27 Causes of DNA Strand Breaks • Reactive Oxygen Species (ROS) generated by • • Errors during cellular processes • • V(D)J recombination, class switch recombination Radiation • • DNA replication, mitosis, meiosis Induced by naturally occurring processes • • metabolic/cellular processes Environmental, medical procedures Chemotherapy • Many chemotherapeutics (etoposide, doxorubicin) inhibit topoisomerase 28 DNA Repair Mechanisms Over 200 genes involved in DNA repair • Major mammalian DNA repair pathways: 1. 2. 3. 4. Nucleotide excision repair Mismatch excision repair Base excision repair DNA strand break repair pathways: Single strand break repair 2. Double strand break repair pathways: 1. Nonhomologous end joining 2. Homologous recombination 1. 29 DNA Repair Mechanisms • Detection of the lesion • • Removal of damaged DNA • • • glycosylases nucleases Repair • • • protein(s) detect and bind DNA lesion DNA polymerase DNA ligase Regulatory proteins • protein kinases to regulate cell cycle (indirect) 30 Nucleotide Excision Repair • Larger mutations distort DNA structure (external damage, e.g., UV damage) • Repair: • • • • Remove damaged section (endonucleases), Fill the gap (DNA polymerase), and Seal the strand (DNA ligase) Disease: Xeroderma pigmentosum (pyrimidine dimers) 31 Xeroderma Pigmentosum (XP) • Rare genetic disease caused by recessively inherited defects in different NER genes • • XP proteins required for NER of UV damage are defective Accumulation of mutations  skin cancer • Patients have increased sensitivity to sunlight • Presents in infancy/early childhood • Severe sunburn, freckles, ulcerative lesions 32 Mismatch Excision Repair • Incorrect pairing of bases during synthesis • Proofreading enzymes correct errors made during replication • • DNA polymerase has 3’ – 5’ exonuclease activity which recognizes mismatched bases and excises them If errors slip through proofreading: In bacteria, methyl-directed mismatch repair finds these errors on newly synthesized strands and corrects them • In euks, mismatch repair finds these errors on newly synthesized strands (more complicated means to distinguish new strand vs old strand) and corrects them • 33 Methyl-directed mismatch repair in E. coli • MMR occurs within minutes of replication • MMR mediated by Mut proteins • Homologous proteins in humans • Mut S recognizes mismatch and recruits Mut L  complex activates Mut H  cleaves daughter strand • Mismatched strand identification based on methylation • • • GATC sequences are methylated on the A residue by DNA adenine methylase Methylated parent strand is correct so daughter strand gets repaired Mismatch identified  endonuclease nicks DNA  DNA removed by exonuclease  gap filled by DNA pol  sealed by DNA ligase 34 Base Excision Repair • Repairs DNA bases damaged by: 1. Alkylation Deamination 3. Oxidation 4. Lost bases (abasic sites) 2. 35 Base Excision Repair Repair: • Remove altered base (glycosylase), • Remove the phosphoribose backbone (AP endonuclease, deoxyribose phosphate lyase) • Fill in the gap (DNA polymerase), and • Seal the strand (DNA ligase) 36 DNA Double Strand Break Repair • DSBs can cause deletions, translocations and fusions  genomic rearrangements • Two pathways for repairing DSBs Homologous Recombination (HR) 1. • • Requires sister chromatid S and G2 phase Non-homologous end-joining (NHEJ) 2. • Post mitotic cells and cycling cells in G1 37 Homologous Recombination 38 Non-homologous End-Joining (NHEJ) Repair • Template is not used • Broken ends are directly ligated • Ku binds to the double stranded break and recruits necessary polymerase and ligase to repair the break 39 Summary • During replication, each of the two parental strands of DNA serves as a template for the synthesis of a complementary strand. • The site at which replication begins is the origin of replication and the site at which replication is occurring is called the replication fork. • Helicases and topoisomerases are required to unwind the DNA helix of the parental strands. • DNA polymerase reads the parental template strand in the 3′-to-5′ direction, producing new strands in a 5′-to-3′ direction. • The precursors for replication are deoxyribonucleotide triphosphates. • As DNA synthesis proceeds in the 5′-to-3′ direction, one parental strand is synthesized continuously, whereas the other exhibits discontinuous synthesis, creating small fragments named Okazaki fragments which are subsequently joined. This is necessary because DNA polymerase can only synthesize DNA in the 5′-to-3′ direction. • DNA polymerase requires a free 3′-hydroxyl group of a nucleotide primer in order to replicate DNA. The primer is synthesized by the enzyme primase, which provides an RNA primer. 40 Summary • The enzyme telomerase synthesizes the ends of linear chromosomes (telomeres). • Errors during replication can lead to mutations, so error checking and repair systems function to maintain the integrity of the genome. • Most DNA damage is detected and repaired by the cell. • Mismatch repair enzymes recognize mis-incorporated bases, remove them from DNA, and replace them with the correct bases. • In nucleotide excision repair, enzymes remove incorrect bases with a few surrounding bases, which are replaced with the correct bases with the help of a DNA polymerase and the template DNA. • The two major DSB repair pathways are DNA non-homologous end-joining (NHEJ) and homologous recombination (HR). 41