DNA Structure and Replication.pdf

DNA Structure and 1 Replication Presented by Dr. Kherie Rowe Assistant Professor of Biochemistry [email protected] Reading: Lippincott Reviews in Biochemistry, 8th Ed., Chapter 30 Meier-Gorlin syndrome can be caused by mutations in one of several genes. Each of thes...

DNA Structure and 1 Replication Presented by Dr. Kherie Rowe Assistant Professor of Biochemistry [email protected] Reading: Lippincott Reviews in Biochemistry, 8th Ed., Chapter 30 Meier-Gorlin syndrome can be caused by mutations in one of several genes. Each of these genes, ORC1, ORC4, ORC6, CDT1, and CDC6, provides instructions for making one of a group of proteins known as the pre-replication complex. This complex regulates initiation of the copying (replication) of DNA before cells divide. 1  Learning Objectives 1. Describe the general structure of the human genome. 2. Give function and the relative proportions of 5 important types of DNA sequences. (centromeric DNA, repetitive DNA (3 forms), telomeres, transcribed region, regulatory DNA). 3. Describe the concept of transposable DNA and its role in genome diversity and formation of pseudogenes. 4. Describe the basic structure of DNA and differentiate it from RNA. 5. Review the structures of nucleotides and the roles of the base, 3’ hydroxyl and 5’ phosphate. 6. Explain the functions of DNA polymerases α, δ, and ε, helicase, beta sliding clamp, DNA ligase, and topoisomerase types I and II in DNA replication. 7. Explain why DNA synthesis occurs only in a 5’ to 3’ direction. 8. Explain how DNA polymerases proofread their product. 9. Describe the complex for initiation of replication and its relationship to cell cycle control. 10. Explain the production and maintenance of telomeres. 2 2 Why Study DNA In all living things, DNA is essential for inheritance, coding for proteins, and providing instructions for life and its processes. DNA dictates how a human or animal develops and reproduces, and eventually dies. DNA is of primary importance to studying the genetic causes of disease , design of gene therapies, and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for determining paternity. 3 3  The Human Genome DNA is the genetic material containing all the information essential for life and the basis for heredity. The human genome is divided into 46 DNA molecules, or chromosomes, consisting of pairs of chromosomes 1 to 22 (autosomes), numbered sequentially according to their size, and of two sex chromosomes that determine whether an individual is male or female. Together, these molecules contain over 6 billion bases that when joined would measure ∼2 meters in length. Thus, the human genome must be extensively packaged in order to fit inside the nucleus, the size of which is in the μm range. Microbiology and Molecular Biology Reviews Jul 2015, 79 (3) 347-372 4 4 Overview: Packaging DNA in Nucleus Length of nuclear DNA: ~ 3 Billion Base Pairs If unwound, it would be ~ 2 meters long So, it is wound to pack in the nucleus 5 5  Two each Two each Two each Two each of of of of Histones H2A H2B H3 H4 Histone Octamer – made up of 2 of: H2A H2B H3 H4 146 bp of DNA Makes 8 (octamer) 6 6 Packaging Nuclear DNA Supercoiling DNA around histone proteins enables it to pack more tightly. These histone proteins work as a spool 7 7  Chromatin DNA can be a relaxed, 11 nm Add histone H1: condensed into a 30 nm fiber fiber Like beads on a string H1 is like a general Exposed to nuclease digestion Protected from nuclease digestion Transcriptionally ACTIVE Transcriptionally LESS ACTIVE POLYNUCLEOSOME 8 The linker histone H1 binds to the entry/exit sites of DNA on the surface of the nucleosomal core particle and completes the nucleosome. It influences the nucleosomal repeat length (NRL) 2 and is required to stabilize higher-order chromatin structures such as the so-called 30-nm fibre. 8  Euchromatin & Heterochromatin More gene expression Less gene expression Increased coiling ->>>>> (Nucleolus = site of rRNA transcription) Dark areas = heterochromatin Histone acetylation / phosphorylation = loses some positive charge = unwinds from DNA = increased transcription Methylation of Cytosine – more packing Topoisomerases can change degree of coiling. 9 9 Video to help you visualize condensation of chromatin is found at https://www.youtube.com/watch?v=gbSIBhFwQ4s 10 10 The Cell Cycle 11 11  Sequences within the Human Genome 1. Composition of the genome a. 27% of the genome is transcribed into mRNAs that will be matured and translated into proteins i. 26% of the genome is introns ii. Only 1.5% of the genome is used to encode proteins b. 20% of the genome is Long Interspersed Elements (LINE) (named L#; L1 is the most abundant at 13% of the genome) c. 13% of the genome is Short Interspersed Elements (SINE) (varied naming; Alu is the most abundant at 7% of the genome) d. 11% are transposons (movable “jumping” genes) e. 8% are heterochromatin (centromeres, telomeres, etc.) 12 An Alu element (or simply, “Alu”) is a transposable element, also known as a “jumping gene.” Transposable elements are rare sequences of DNA that can move (or transpose) themselves to new positions within the genome of a single cell. Alu elements are about 300 bases long and are found throughout the human genome. A telomere is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Telomeres are a widespread genetic feature most commonly found in eukaryotes. The centromere links a pair of sister chromatids together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm and a long arm on the chromatids. During mitosis, spindle fibers attach to the centromere via the kinetochore. 12  Sequences Within The Human Genome 2. Transposable regions may cause genomic changes when they are inserted or excised a. Increase or decrease the spacing between regulatory units from genes (changing expression) b. Insert or delete protein coding regions changing exons (may alter protein function, localization or regulation) c. Alter gene expression by creating pseudogenes (non-functional gene copies or non-expressed copies) Clinical Correlation: Insertion of L1 into the clotting factor VIII gene caused hemophilia (Kazazian et al., 1988). Similarly, L1 was found to be present in the APC (Adenomatous Polyposis Coli) genes in colon cancer cells but not in the APC genes in healthy cells in the same individuals. Front. Neurol., 20 August 2019 | https://doi.org/10.3389/fneur.2019.00894 13 13  Sequences Within The Human Genome 3. Repetitive DNA a. Promotes gene repair by using a copy from another chromosome after double strand breaks b. Allows gene duplication by misalignment at a repeat during recombination or repair c. Allows gene deletion by misalignment of repeats during recombination or repair 14 14  Example of Repetitive DNA Centromeres are required for normal chromosome segregation in mitosis and meiosis. The primary sequence at human centromeres is a satellite DNA (171 bp monomers) organized in a tandem head-to tail fashion. The monomeric sequences have 50-70% homology. A set number of monomers give rise to a higher order repeat and confer chromosome-specificity. Higher order repeats are themselves reiterated hundreds to thousands of times. 15 15  Sequences WITHIN THE Human Genome 16 Minisatellites and their shorter cousins, the microsatellites, together are classified as VNTR (variable number of tandem repeats) DNA. Confusingly, minisatellites are often referred to as VNTRs, and microsatellites are often referred to as short tandem repeats (STRs) or simple sequence repeats (SSRs). The nucleotide sequence of the repeats is fairly well conserved across species. However, variation in the length of the repeat is common. For example, minisatellite DNA is a short region (1-5kb) of repeating elements with length >9 nucleotides. Whereas microsatellites in DNA sequences are considered to have a length of 1-8 nucleotides. The difference in how many of the repeats is present in the region (length of the region) is the basis for DNA fingerprinting. 16 Central Dogma of Molecular Biology 17 17 Structure of Nucleotides 18 18 Bases in Nucleotides 19 19 20 20 Phosphodiester linkages in the covalent backbone of DNA and RNA 21 21 Hydrogen Bonding Between Complementary DNA Strands Chargaff’s Rule: “In double stranded DNA, the number of A’s is equal to the number of T’s, and the number of G’s is equal to the number of C’s”. The bases are complementary to each other, one purine with one pyrimidine. 22 22 Structure of Double-Stranded DNA (dsDNA) The strands in double-stranded DNA are Antiparallel Complementary Bases are connected via hydrogen bonds 2 H bonds connect A with T (and T with A) 3 H bonds connect C with G (and G with C) Number of Purines = Number of Pyrimidines Nucleotides are connected in backbone via phosphodiester bonds ‘Backbone’ is sugars and phosphates: they are invariant Bases are internal: they vary 23 23  Right-Handed, Antiparallel Double Helix (B-DNA) Double helix ~ 3.4 Å/base The hydrophilic phosphodeoxyribose backbones face out The bases are found on the inside. 10.5 bases per turn (36 Å) Minor and Major grooves provide binding sites for regulatory proteins Anti-tumor drug cisplatin 24 Cisplatin binds to the N7 reactive center on purine residues and as such can cause deoxyribonucleic acid (DNA) damage in cancer cells, blocking cell division and resulting in apoptotic cell death. The 1,2-intrastrand cross-links of purine bases with cisplatin are the most notable among the changes in DNA. 24 DNA duplex can exist in different 3-D forms: B-form: The Watson-Click structure, standard form, is the most stable under physiological conditions A-form: A dehydrated form of B Z-form : Left-handed; GC rich sequences; Short Z-DNA tracts may play a role in gene regulation. 26 26 DNA Synthesis Reaction catalyzed by DNA polymerases: Covalent extension of a DNA primer strand in the 5’ → 3’ direc on. In this example, the existing chain terminates at the 3’ end with the nucleotide deoxyguanosine-5- phosphate (deoxyguanylate). DNA polymerase-catalyzed addition of deoxythymidine monophosphate from the precursor deoxythymidine triphosphate (dTTP) to the 3’ end of the chain with the release of pyrophosphate. 27 27 DNA Synthesis All polymerases require preexisting DNA with two essential components, one providing a primer function and the other a template function: 1. The primer DNA provides a terminus with a free 3’-OH to which nucleotides are added during DNA synthesis. *AZT: Antiretroviral medication used to 2. The template DNA provides the prevent and treat nucleotide sequence that specifies HIV/AIDS the complementary sequence of the growing DNA chain. 28 28 DNA Replication DNA replication is semi-conservative. A new DNA strand (red) is always synthesized in the 5 → 3 direction. The template is read in the opposite direc on, 3 → 5. The leading strand is continuously synthesized in the direction taken by the replication fork. The other strand, the lagging strand, is synthesized discontinuously in short pieces (Okazaki fragments) in a direction opposite to that in which the replication fork moves. The Okazaki fragments are spliced together by DNA ligase. In bacteria, Okazaki fragments are ~1,000 to 2,000 nucleotides long. In eukaryotic cells, they are 150 to 200 nucleotides long. 29 29 Fidelity of DNA Replication DNA replication has evolved a mechanism for proofreading the nascent DNA chain as it is being synthesized. This involves scanning the termini of nascent DNA chains for errors and correcting them. This process is carried out by the 3’ → 5’ exonuclease activities of DNA polymerases. 30 30 Eukaryotic DNA Replication   31 31 Prokaryotic DNA Replication Components DNA Pol III DNA Pol I replaces RNA primer with DNA DNA Pol III 32 32 Initiation of Replication At the G1 , a cell checks whether internal and external conditions are right for division. Some of the factors a cell might assess: Size Nutrients Molecular signals DNA integrity If a cell doesn’t get the go- ahead cues it needs at the G1 checkpoint, it may leave the cell cycle and enter a resting state called G0 phase. 33 33  Regula on of G1 → S Phase Transi on Early G1, retinoblastoma protein (pRb) binds and sequesters transcription factor E2F, preventing cell cycle progression In the G1/S phase, pRb is phosphorylated. pRb Phosphorylation breaks up the pRB-E2F complex o E2F activates transcription of genes involved in S phase o E2F transcribes genes for DNA polymerase, & Cyclin E 34 34  Initiation of Replication DNA replication is initiated at specific sites in the genome known as the ‘origins’ which are recognized and bound by origin binding proteins. Replication commences at a single origin in prokaryotes and at multiple origins in eukaryotes. However, the basic mechanism of replication is conserved. In eukaryotes, initiator proteins ORC, Cdc6 and Cdt1 recruit the replicative helicase. The eukaryotic replicative helicase is a complex of proteins called the CMG helicase consisting of Cdc45, Mcm2-7 and GINS protein complex. 35 35  Regulation of CDC6 CDC6 is normally present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is largely transcribed during G1 phase. On the onset of the S phase, CDC6 is phosphorylated and inactivated by Cdk2 (the Cdc28-Clb5-Clb6 complex). The inactivated CDC6 is then targeted for degradation by SCFCDC4-dependent ubiquitinylation and afterwards degraded by the proteasome. Thus, the regulation of CDC6 is tightly correlated to the activity of Cdk2. Two states can be distinguished. In the first state (during G1 phase) Cdk2-activity is low, CDC6 can accumulate, hence the pre-RC can be formed but not activated. In the second state, Cdk2-activity is high, CDC6 becomes inactivated, hence the pre-RC is activated but not formed. This change assures that DNA replication is performed only once per cell cycle. Regulation of CDC6 is one of several redundant mechanisms that prevent re-replication of the DNA in eukaryotic cells. 36 The assembly of the pre-replicative complex (pre-RC) at origins during G1 phase is called ‘origin licensing’ The helicase is inactive in the pre-RC and is activated only in the S phase when origins ‘fire’ due to the activity of CDK/DDK kinases. Once origins fire, DNA synthesis begins, and the initiator proteins are exported out of the nucleus and/or degraded to prevent re-replication. 36 Eukaryotic Protein Function ORC proteins Recognition of origin of replication Topoisomerase I/II Relieves positive supercoils ahead of replication fork Mcm DNA helicase that unwinds parental duplex Cdc6, Cdt1 Loads helicase onto DNA RPA/SSB Maintains DNA in single‐stranded state RFC Subunits of the DNA polymerase holoenzyme that load the clamp onto the DNA pol / Primary replicating enzymes; synthesize entire leading strand and Okazaki fragments; have proofreading capability PCNA Ring‐shaped subunit of DNA polymerase holoenzyme that clamps replicating polymerase to DNA; works with pol III in E. coli and pol  or  in eukaryotes Primase Synthesizes RNA primers pol  Synthesizes short DNA oligonucleotides as part of RNA–DNA primer DNA ligase Seals Okazaki fragments into continuous strand FEN-1/RNase H Removes RNA primers; pol I of E. coli also fills gap with DNA 37 37  Replication Termination At the end of the lagging strand, it is impossible to attach an RNA primer, meaning that there will be a small amount of DNA lost each time the cell divides. In order to prevent this, telomeres are repeated hundreds to thousands of times at the end of the chromosomes. The telomeres of humans contain a tandemly repeated sequence TTAGGG. The synthesis of telomeres is mainly achieved by the cellular reverse transcriptase telomerase, an RNA- dependent DNA polymerase that adds telomeric DNA to telomeres. 38 38  Telomeres Telomeres maintain genomic integrity in normal cells, and their progressive shortening during successive cell divisions induces chromosomal instability which leads to cell senescence, where the cell is unable to divide, or apoptotic cell death. Clinical Correlation - In most cancer cells, telomere length is maintained by telomerase. → telomere length and telomerase activity are crucial for cancer initiation and the survival Healthy telomeres are curled into a ‘loop’ of tumors. structure at the ends of our chromosomes. N Engl J Med 2009; 361:2353-2365 DOI: 10.1056/NEJMra0903373 39 39  Some Key Points on DNA Replication DNA replication is complex, requiring the participation of a large number of proteins. DNA synthesis is continuous on the progeny strand that is being extended in the overall 5’ → 3’ direc on but is discon nuous on the strand growing in the overall 3’→5’ direction. New DNA chains are initiated by short RNA primers synthesized by DNA primase. DNA synthesis is catalyzed by enzymes called DNA polymerases. All DNA polymerases require a primer strand, which is extended, and a template strand, which is copied. All DNA polymerases have an absolute requirement for a free 3’-OH on the primer strand, and all DNA synthesis occurs in the 5’ to 3’ direction. The 3’→ 5’ exonuclease ac vi es of DNA polymerases proofread nascent strands as they are synthesized, removing any mispaired nucleotides at the 3’ termini of primer strands. The enzymes and DNA-binding proteins involved in replication assemble into a replisome at each replication fork and act in concert as the fork moves along the parental DNA molecule. 40 40