DNA Replication

• Prokaryotes do not have histones.
• Eukaryotic cells' chromatin consists of DNA complexed with histones in nucleosomes.
• Histones are basic proteins with high arginine and lysine content.
• Eight histone molecules form an octamer, which winds about 140 base pairs of DNA to form a nucleosome core.
• Histone H1 complexes with the DNA that joins one nucleosome core to the next.
• Base pairing rules dictate DNA strands are complementary.
• The two strands of the DNA double helix run antiparallel, meaning their chemical orientations differ.
• Conventions dictate that a base sequence is usually written with the 5' P terminal end on the left.

DNA Replication Models

• Strands can separate and act as templates for new complementary strand formation.
• In conservative replication, parental DNA strands remain together and newly synthesized DNA strands go to the other daughter cell.
• In semiconservative replication, each daughter cell receives one parental DNA strand and one newly synthesized complementary strand using the parental strand as a template.
• Matthew Stanley Meselson and Franklin Stahl proved in 1957 that DNA replicates semi-conservatively.

Introduction to DNA Replication

• DNA replication is the process of copying DNA before cell division.
• In the semi-conservative model, each new DNA molecule consists of one parental and one newly synthesized strand.
• DNA replication in eukaryotes occurs during the S-phase of the cell cycle.

Basic Requirements for DNA Synthesis

• Four deoxynucleotide triphosphates (dNTPs) are the substrates of DNA synthesis.
• The four dNTPs are deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP).
• Template directs complementary nucleotide addition.
• Each parental DNA strand serves as a template in semiconservative replication.
• A primer prepares the template strand for nucleotide addition.
• New nucleotides are attached to the primer's 3'-end, so the new synthesis occurs in a 5' to 3' direction.
• DNA-dependent DNA polymerases are the enzymes involved.

Enzyme Activities in Replication

• DNA Polymerase synthesizes DNA in the 5' to 3' direction, like DNA Pol III in bacteria and DNA Pol δ in eukaryotes.
• Exonuclease proofreading corrects errors in the 3' to 5' direction, like DNA Pol I and III in bacteria and DNA Pol δ and ε in eukaryotes.
• Exonuclease removes primers or DNA fragments in the 5' to 3' direction, like DNA Pol I in bacteria.

Characteristics of Prokaryotic DNA Replication

• Prokaryotes feature a single, circular chromosome.
• Replication typically originates from a single origin known as OriC.
• Replication occurs bidirectionally from the origin at approximately 1000 nucleotides per second.

Initiation in Prokaryotes

• DnaA protein binds to OriC, causing localized DNA unwinding.
• DnaB (Helicase) unwinds the DNA helix ahead of the replication fork.
• Single-strand binding proteins (SSBs) stabilize unwound single-stranded DNA.
• DNA gyrase (Topoisomerase II) alleviates supercoiling tension ahead of the replication fork.
• Primase (DnaG) synthesizes short RNA primers to provide starting points for DNA synthesis.

Elongation in Prokaryotes

• DNA Polymerase III synthesizes the leading strand continuously in the 5' to 3' direction.
• Discontinuous synthesis on the lagging strand creates Okazaki fragments.
• DNA Polymerase III extends RNA primers with DNA nucleotides.
• DNA Polymerase I removes RNA primers and replaces them with DNA nucleotides.
• DNA ligase seals the nicks between Okazaki fragments, forming a continuous DNA strand.

Termination in Prokaryotes

• Specific sequences called Ter sites signal the end of replication.
• Tus proteins bind to Ter sites, which inhibits helicase activity and halts replication.
• Topoisomerase IV decatenates interlinked daughter chromosomes to ensure proper segregation.

Characteristics of Eukaryotic DNA Replication

• Eukaryotic DNA replication involves multiple linear chromosomes.
• Replication starts at multiple origins along each chromosome.
• The replication rate is approximately 50 nucleotides per second.
• Cell cycle checkpoints tightly regulate replication to ensure accuracy.

Initiation in Eukaryotes

• The Origin Recognition Complex (ORC) binds to replication origins, marking them for initiation.
• MCM Helicase unwinds the DNA double helix at the replication fork.
• Replication Protein A (RPA) stabilizes single-stranded DNA regions.
• Topoisomerases I & II relieve supercoiling tension ahead of the replication fork.
• DNA Primase-Polymerase complex synthesizes RNA primers and initiates DNA synthesis.

Primosome

• The combination of prepriming complex and primase.
• Prepriming complex forms prior to RNA primer synthesis of several proteins.
• It binds the single strand of DNA, displacing some single-stranded DNA-binding proteins.
• Prepriming complex displaces SSB in an ATP-dependent process.
• This initiates Okazaki fragment formation by moving along the lagging strand in the 5' to 3' direction.

Elongation in Eukaryotes

• DNA Polymerase carries out leading strand synthesis.
• DNA Polymerase carries out lagging strand synthesis, producing Okazaki fragments.
• RNase H removes RNA primers in primer removal and replacement.
• FEN1 (Flap Endonuclease 1) processes the 5' ends of Okazaki fragments.
• DNA Polymerase fills in gaps left after primer removal.
• DNA Ligase I joins Okazaki fragments to form a continuous DNA strand.

Topoisomerase

• Topoisomerase alters DNA supercoiling by making transient single-strand breaks (Type I) or double-strand breaks.
• Type IA Topoisomerases relax only negatively supercoiled DNA by changing the linking number.
• Type IB Topoisomerases relax negative and positive supercoils by a controlled rotation mechanism in eukaryotic cells.
• Type II DNA Topoisomerases bind tightly to the DNA double helix and make transient breaks in both strands.
• The resealing transient break relieves negative and positive supercoils and is ATP-dependent.
• Fluoroquinolones inhibit Top 2 in prokaryotes.
• Etoposide/Tenoposide inhibit Top 2 and are used in cancer cells in Eukaryotes.

Termination in Eukaryotes

• End Replication Problem: DNA polymerases cannot fully replicate the 3' ends of linear chromosomes.
• Telomerase, a ribonucleoprotein enzyme, extends telomeres using its RNA component as a template.
• The Shelterin Complex binds to telomeres, which protects them from degradation and prevents recognition as DNA damage.
• Telomeres are repetitive DNA sequences (TTAGGG in humans) at the ends of eukaryotic chromosomes.
• Telomeres act as protective caps, preventing the loss of essential genetic information during DNA replication.

Telomeres

• The prevention of genetic loss is functions of telomeres.
• DNA polymerase cannot fully replicate the 3' ends of linear chromosomes due to the end-replication problem, which leads to gene loss over time without telomeres.
• Chromosome Protection: Telomeres prevent chromosomes from fusing with each other, which could cause genome instability.
• The Shelterin complex (TRF1, TRF2, POT1, TIN2, RAP1, and TPP1) binds to telomeres and shields them from degradation.
• Regulation of Cellular Lifespan: Telomere shortening acts as a biological clock for cellular aging (senescence).
• Cells undergo apoptosis or permanent cell cycle arrest (replicative senescence) when telomeres become critically short.
• Cancer and Immortality: Cancer cells reactivate telomerase, which allows them to divide indefinitely.
• Most cancers exhibit upregulated telomerase activity, making telomerase a target for cancer therapies.

Checkpoints Ensuring Accurate Replication

• S-Phase Checkpoint ensures DNA damage is repaired before replication continues.
• ATR & ATM Kinases detect stalled replication forks and activate repair pathways.
• G2/M Checkpoint prevents mitosis if replication is incomplete.
• p53 & Rb Proteins halt cell cycle progression if errors are detected.
• Mismatch Repair System (MMR) detects and corrects base-pair mismatches after replication.
• MLH1, MSH2, and MSH6 are involved in the Mismatch Repair System.

Diseases Associated with DNA Replication Errors

• Cancer (Uncontrolled Cell Division): p53 mutations prevent apoptosis in cells with replication errors, leading to cancer development.
• Defective DNA polymerases can cause excessive mutations, which contributes to colorectal cancer.
• Bloom Syndrome: A defective RecQ Helicase (BLM gene mutation) leads to increased recombination and chromosomal instability.
• Symptoms include short stature, cancer predisposition, and sun sensitivity.
• Werner Syndrome: Defective WRN Helicase is involved in telomere maintenance, causes premature aging, cardiovascular disease, and increased cancer risk.
• Lynch Syndrome (HNPCC - Hereditary Non-Polyposis Colorectal Cancer): Defective Mismatch Repair (MMR) Proteins (MLH1, MSH2, MSH6) leads to microsatellite instability and increased risk of cancers.
• Xeroderma Pigmentosum (XP): Defective Nucleotide Excision Repair (NER) enzymes (XPA, XPC, XPV, etc.) cause an inability to repair UV-induced thymine dimers, leading to extreme sunlight sensitivity and early-onset skin cancers.
• Seckel Syndrome: A defect in ATR kinase monitors replication stress and causes growth retardation, microcephaly, and developmental delays.
• Fanconi Anemia: This means there is are defective DNA Interstrand Crosslink Repairs that result in bone marrow failure, leukemia predisposition, and developmental abnormalities.

Key Points on DNA Replication

• Prokaryotic DNA Replication: A simple, fast, single-origin process involving DnaA, DnaB, and Pol III.
• This is complex, multiple origins, regulated by ORC, Pol a, Pol d, and e.
• Telomerase action resolves the End-Replication Problem and is critical for chromosome stability.
• Checkpoints Ensure Accuracy: ATR/ATM kinases, p53, and CDKs regulate replication.
• Replication Defects Lead to Disease: Resulting in cancers, aging syndromes, and genomic instability disorders.

Key Features of Mitochondrial DNA Replication

• The mitochondrial DNA replication contains circular, double-stranded DNA (~16.5 kb in humans).
• Replication contains multiple Origins of Replication (OriH and OriL).
• Mitochondria are independent of Cell-Cycle Regulation.
• A minimal error repair mechanisms which leads to higher mutation rates.

Regulation/Proteins in Mitochondrial DNA Replication

• DNA Polymerase y (Pol γ): Main polymerase, responsible for mtDNA synthesis.
• Twinkle Helicase unwinds mitochondrial DNA.
• mtSSB (Mitochondrial Single-Stranded Binding Protein) stabilizes single-stranded DNA.
• PrimPol provides RNA primers for DNA Pol y.
• TFAM (Mitochondrial Transcription Factor A) regulates mtDNA replication.
• RNase H1 removes RNA primers.
• DNA Ligase III seals Okazaki fragments on the lagging strand.
• Topoisomerase 3a resolves supercoiling during mtDNA replication.

Overview of Transcription

• Template: DNA (antisense strand)
• Template: RNA is transcribed from the antisense strand
• Product: RNA (other non-coding RNAs)
• RNA synthesis happens in the 5' to 3' direction, meaning nucleotides are added to the 3' end of the growing RNA chain.
• Enzyme: The RNA polymerase that catalyzes the reaction.
• Regulation: Is controlled by promoter sequences, transcription factors, enhancers, and repressors.

Initiation in Prokaryotes

• The process happens at a promoter, a specific DNA sequence that RNA polymerase identifies with the holoenzyme:
• Core enzyme -> responsible for RNA synthesis.
• Sigma factor -> helps RNA polymerase bind correctly.
• Promoter regions have: -35 sequence (TTGACA) - Recognized by RNA polymerase binding, Pribnow box (-10 sequence, TATAAT) - Important for initiation.
• Once RNA polymerase binds to the promoter, it unwinds a small part to form the bubble(~17 base pairs.)
• The first nucleotides of RNA are synthesized, and the sigma factor is released when a hybrid is formed, allowing elongation.

Elongation During Transcription

• During the elongation phase, RNA polymerase moves along the DNA and adds ribonucleotides template strand.
• The enzyme does not need a primer.
• The RNA chain grows in the 5' to 3' direction.

Termination During Transcription

• Termination has two mechanisms in prokaryotes
• Rho-independent termination: This happens when the RNA forms a hairpin loop followed by a series of uracils (U-rich sequence).
• The weak A-U bonds between RNA and DNA causes the RNA to detach, ending transcription.
• Rho-dependent termination: Happens with the Rho protein, an ATP-dependent helicase in which The Rho binding site (Rut site) on binds and moves towards the RNA polymerase
• When Rho catches up with the RNA , it unwinds the RNA-DNA pause transcript.

Eukaryotic Transcription - RNA Polymerases

• Eukaryotes have three major RNA polymerases: RNA Polymerase I → Synthesizes rRNA (except 5S rRNA), RNA Polymerase II → Synthesizes mRNA in the nucleus, RNA Polymerase III → Synthesizes rRNA, and other small RNAs.

Transcription - Initiation in Eukaryotes

• Eukaryotic promoters are very complex. Core promoter: TATA box (~ -25 bp for part of the TFIID complex), Initiator sequence (Inr) (~ +1 position) - Overlaps the initiation site
• Enhancers modulate transcription rate and GTFs ( e.g., TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH) help RNA polymerase II binding, TFIIH is has helicase activity which initates transcription.

Transcription - Elongation and Termination in Eukaryotes

• RNA polymerase proceeds along the DNA and synthesizes RNA in the 5' to 3' direction, and elongation factors and topoisomerases are involved.
• There less understand and vary by polymerase type. RNA Polymerase III and 3' End Processing happens termination factor similar to Rho-independent termination, RNA Polymerase III: Terminates at a poly sequence similar to prokaryotic Rho-independent termination.
• RNA Polymerase II: Degrades with an exonuclease

Post Transcription in Eukaryotes

• Eukaryotic Modifications must occur to become mature like: 1. 5' Capping: adds a 7-methylguanosine at the 5' end, RNA initiation, 2. Splicing removes joins exons, alternative splicing, 3. 3' Polyadenylation: Increase stability

Disesases Involving Transcript Dysregulation

• Diseases: Mutatations in transcription factors, Huntington's Disease, Beta-Thalassemia - Mutation in -globin gene affecting transcription regulation., Lupus (SLE): Autoantibodies against snRNPs affecting splicing.

Transfer RNA

• Distinct regions: Acceptor Stem (End): 5' end starts with a G and CCAA sequence and synthetase.
• The Anticodon Arm has triplet code in tranlation and specificity, modified
• Nucleosides and Tertiary Structure

tRNA and Enzymes

• RNA transcipts synthesis by RNA Polymerase III in the eukaroytes which the primary must modify before becoming mature
• tRNA Nucleotidyltransferase and Ribonuclease P with a ribozyme.

Direct Repair - DNA

• DNA backbone without breaking the DNA that are non-helix with endonuclease.
• End Joining and homolougs Recombination - error free by template.

Test your knowledge of DNA replication. This quiz covers key enzymes, processes like lagging strand synthesis, the role of primers, and differences between prokaryotic and eukaryotic replication. Also covers the structure and organization of DNA.