DNA Damage and Repair & RNA Transcription Lecture Notes PDF

BMS: 141 Lecture No: 5 Title: 1: DNA damage and repair 2: RNA transcription Instructor Name: Dr Lamees Dawood Medicine and Surgery Program Fall 2024  ILOs Intended learning Outcomes (ILOs): Studying this topic should enable you to: 1....

BMS: 141 Lecture No: 5 Title: 1: DNA damage and repair 2: RNA transcription Instructor Name: Dr Lamees Dawood Medicine and Surgery Program Fall 2024  ILOs Intended learning Outcomes (ILOs): Studying this topic should enable you to: 1. Describe DNA repair systems. 2. Explain the molecular mechanisms of diseases due to failure of repair systems. 3. Compare between different repair systems.  Definition - Identification and correction of damaged DNA How? As DNA molecule consists of two complementary strands. So, damage in one strand can be corrected using the undamaged complementary strand as a template Causes of DNA damage  Causes of DNA damage 1. Replication (copying) errors which escape the proofreading system. These can be repaired by the mismatch repair system. 2- Environmental insults by DNA exposure to: a-Chemicals e.g nitrous oxide, deaminates Cytosine into Uracil, Adenine into Hypoxanthine (HX) & Guanine into Xanthine. This type of damage is repaired by base excision repair. b- Radiation as UV produces thymine dimer (T=T) which is repaired by nucleotide excision repair. 3. Bases may be also altered or lost spontaneously from mammalian DNA at a rate of 10,000 purines per cell per day and this damage is repaired by base excision repair. Why do we need DNA damage repair systems? If the DNA damage is not repaired, a permanent mutation may result that can lead to a number of serious effects.  Overview of repair Most of the repair systems include the following common steps: 1. Recognition of the damage (lesion) on the DNA. 2. Removal (excision) of the damage. 3. Replacement (filling the gap) using the sister strand as a template for DNA synthesis by DNA polymerase. 4. Ligation.  I- Strand directed mismatch repair: A) Steps of repair in Prokaryotes : 1- Identification of the mismatched strand: A group of proteins called Mut proteins (S, H, L) form a complex with each other: Mut S identifies the mismatch, Mut H finds GATC sequences on unmethylated DNA strand, Mut L links Mut S to Mut H. 2- One of Mut proteins (H) has an endonuclease activity and nicks the mismatched strand on the 5` side of the G in GATC sequence 3- A complex protein consists of DNA helicase and an exonuclease then degrades the DNA strand from the nick point toward the mismatch. 4- The gap left is filled, using the sister strand as a template, by DNA polymerase III enzyme and the DNA is sealed by the Ligase enzyme.  Complete Template strand of DNA is --- while the newly synthesized strand is not -------protein recognizes and binds to the mismatched base pair. -------protein binds to mut S coordinating the cleaves and excision Mut H protein binds to methylated ---- sequence on the parent strand for ‫ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ‬repair. B-Steps of repair in eukaryotes -Eukaryotes have a similar mismatch repair system, but the mechanism by which they discriminate between old and newly replicated DNA strand is not known (not by methylation as in E. coli). -Clinical importance: Mutation in the proteins involved in mismatch repair system in human results in Hereditary Nonpolyposis Colorectal Cancer (HNPCC).  II. Base excision repair Steps: a- DNA glycosylase removes the deaminated base by cleaving the N-glycosidic bond to create an AP site i.e. Apyrimidinic or Apurinic site (without pyrimidine or purine base) b) AP endonucleases: cut the internal phosphodiester bond at the 5′-end of the AP site (upstream) creating a free 3'-OH end. c) AP Lyase: removes the single, empty, sugar phosphate residue. d) DNA polymerase β and DNA ligase fill the gap and seal the DNA. III. Nucleotide excision repair  DNA repair in relation to eukaryotic cell cycle: G1 phase: 1. Deamination which converts cytosine to uracil is corrected by base excision repair (uracil gylcosylase). 2. Loss of purine or pyrimidine is corrected by base excision repair (AP endonuclease). 3. UV radiation produces thymine dimers which is corrected by nucleotide excision repair. G2 phase Copying errors during DNA replication are corrected by mismatch repair Clinical significance of repair system Errors repair system in euokryotes could result in disease 1- Xeroderma pigmentosa 2- Hereditary Nonpolyposis Colorectal Cancer (HNPCC).  1- Xeroderma pigmentosa -Rare autosomal recessive disease -Cause: Defect in any of genes required for repair of thymine- thymine dimers (Most common damage caused by exposure to UV irradiation). -Clinical picture: a- Extreme photosensitivity b-High susiptability to skin cancer in sun exposed area 2-Hereditary Nonpolyposis Colorectal Cancer (HNPCC). -Rare autosomal dominant disease -Cause: Defect in any of genes required for repair of mismatched base (mostly MLH1 and MSH2). In class assessment  What is the name of the DNA repair system in E. coli in which dual incisions are made in the damaged part of the double helix, and a 12-13 base segment is removed and replaced with new DNA? a) Mismatch repair b) Base excision repair c) Nucleotide excision repair d) AP site repair c  Which of the following is the name of the human genetic disorder resulting from defects in nucleotide excision repair? a) Hereditary nonpolyposis colorectal cancer (HNPCC) b) Xeroderma pigmentosum (XP) c) Lynch syndrome d) Diabetes b  References for further readings Lippincott Illustrated Review of biochemistry 8th edition Oxford Hand book of Medical Science 2nd edition Clinical Key Student RNA Synthesis (Transcription)  ILOs By the end of this lecture the student should be able to: ▪ Identify the structure of RNA polymerase ▪ Define transcription process. ▪ Explain stages of transcription. ▪ Differentiate the prokaryote and eukaryote transcription TRANSCRIPTION (RNA SYNTHESIS) I Prokaryotes 1- Definition ▪Transcription is defined as synthesis of RNA from DNA. Results in the transfer of the information stored in double stranded DNA into a single stranded RNA, which is used to direct the synthesis of its proteins. 2- Requirements A. Four ribonucleotides building blocks. B. RNApolymerase. C. Promoter. D. Template strand. E. Enhancers.  A. RNA Polymerase DNA dependent RNA polymerase, called RNA polymerase (RNAP) responsible for the synthesis of RNA using DNA template. Prokaryotes have single RNA polymerase that transcribes all three RNAs,i.e. mRNA, t-RNA and r-RNA. RNAP contains four subunits (2α, β,β), which form the core enzyme (Apoenzyme).  A- RNA Polymerase The active enzyme,the holoenzyme contains core enzyme and a fifth subunit called sigma subunit. Which is required for binding of the RNA- polymerase to specific regions (promoter region) of DNA template.  B- Prokaryotic promoters 1.Pribnow box (–10 region) has the nucleotide sequence TATAAT and is usually found 10 base pairs away from (upstream) the start point. 2.The –35 region, has the nucleotide sequence TTGACA. Start point or initiation site. The binding of the RNA polymerase to the DNA template results in the unwinding of the DNA double helix.  B- Prokaryotic promoters ▪The enzyme then catalyzes the formation of phosphodiester bond between the first two ribonucleotides complementary to DNA template sequence. ▪Unlike the initiation of replication, transcriptional initiation does not require a primer.  C- Template strand of DNA ▪This is the strand over which complementary RNA strand will be synthesized. ▪This template strand is also known as antisense strand. ▪The other strand of DNA is known as sense or coding strand  3- Steps of Transcription RNA synthesis involves: 1. Inititiation. 2. Elongation 3. Termination A- Initiation ▪ Initiation of transcription involves the binding of RNA polymerase (core enzyme +σ factor) to the DNA template at the promoter site. ▪The sigma factor enables the RNA polymerase (holoenzyme) to recognize and bind to promoter sequences. Promoters are characteristic sequences (consensus) of DNA which are different in prokaryotes and eukaryotes  Initiation ❑ Describe the process of initiation. RNA POLYMERASE  B- Elongation ▪ Elongation proceeds after the formation of the first phosphodiester bond. ▪ After formation of 10 phosphodiester bonds of the new RNA, sigma (σ) subunit dissociates from the core enzyme. ▪ RNA polymerase utilizes ribonucleotide triphosphates (ATP, GTP, CTP and UTP) for the formation of RNA. ▪ The process of elongation of the RNA chain continues until a termination signal is reached.  C- Termination 1.Rho-dependent Rho-dependent termination, requires a protein factor called rho (ρ) which recognizes the termination signal and has an ATP dependent helicase activity that displaces the RNA polymerase from template resulting in termination of RNA synthesis. 2.Rho-independent (intrinsic ) Formation of a secondary structure (hair-pin loop) in the newly synthesized RNA, which removes the RNA polymerase from DNA template, resulting in the release of the transcript. This is followed by a sequence of four or more uracil residues, which also are essential for termination. C- Termination  4- Inhibitors of Transcription Antibiotics inhibit RNA synthesis of prokaryotes not eukaryotes,For example: A- Rifampin: It is an anti tuberculosis drug, which inhibits the initiation of transcription by binding β-subunit of prokaryotic RNA polymerase. B- Actinomycin D: Dactinomycin is a therapeutic agent in the treatment of some cancer. It binds tightly to double helical DNA and thereby prevents the movement of the RNA polymerase.  II- Transcription in Eukaryotes Promoters Each type of eukaryotic RNA polymerse uses a different promoters. 1- Hogness box or TATA box: It is a stretch of 6 nucleotides and located 30 nucleotides upstream of the transcription starting point. 2.CAAT box: It is stretch of 8 nucleotides and located about 90 nucleotides upstream of the transcription starting point.  Transcription in Eukaryotes Promoters 3. GC box: It is a stretch of 6 nucleotides and is located about 75 nucleotides upstream of the transcription starting point. Eukaryotic RNA polymerase RNA polymerases I, II and III found in nucleus. 1.RNA Polymerase I: It catalyzes the synthesis of ribosomal RNA. 2.RNA Polymerase II: It catalyzes the synthesis of m-RNA and small nuclear RNAs (sn-RNA). 3.RNA Polymerase III: It catalyzes the synthesis of tRNA. Besides the three nuclear RNA polymerases, in eukaryotic cell, a fourth type of RNA polymerase is found in mitochondrial matrix known as mitochondrial RNA polymerase (mtRNAP). Similar to prokaryotic RNA polymerase, mtRNA polymerase catalyzes the synthesis of all the three types of RNA, i.e. mRNA, tRNA and rRNA.  Enhancers Short region of DNA that can increase transcription of genes can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or thousands of nucleotides away. When a DNA -binding protein binds to the enhancer, the shape of the DNA changes, which allows interactions between the activators and transcription factors to occur.  Eukaryotic initiation Initiation starts by recognizing and binding of TFIID to TATA box Attachment of several transcription factors with RNA polymerase II forming preinitiation complex. These transcription factors named TFII (A,B,D,E,F and H) The most important is D which recognize TATA box and H which having helicase like activity. For starting transcription RNA polymerase which is loaded by many TRFs is released by hydrolysis of ATP  Eukaryotic Elongation Polymerase moves downstream with unwinding the DNA and elongating the RNA transcript in`5 to`3 using the ribonucleotides ATP,GTP,CTP and UTP with base pairing role. Pyrophosphate is released when each new ribonucleotide is added.  Eukaryotic Termination RNA Polymerase I and III termination by rho factor or intrinsic (rho independent). RNA Polymerase II transcript a pre-mRNA tail about 1000 nucleotides from the template after gene transcription ,this help termination of RNA transcript. Eukaryotic inhibition Inhibitors of RNA polymerase II: This enzyme is inhibited by 1- α-amanitin—a potent toxin produced by the poisonous mushroom (sometimes called “death cap”)forms a tight complex with the polymerase. 2- Rifampicin inhibits beta subunit of RNA polymerase II 3- Acridine orange and Actinomycin D is used to block elongation. In Class Assessment  Questions Q1:Which of these subunits of RNA polymerase is totally required to initiate transcription? (a)alpha (α) (b)sigma (σ) (c)omega (ω) (d)beta (β) Q2 Synthesis of RNA frm a DNA template is known as: A)Replication B)Translation C)Transcripition D)Mutation Q3 Prokaryotic DNA –dependent RNA polymerase is a : A)Monomer B)Dimer C)Trimer D)Tetramer  Questions Q1:Which of these subunits of RNA polymerase is totally required to initiate transcription? (a)alpha (α) (b)sigma (σ) (c)omega (ω) (d)beta (β) Q2 Synthesis of RNA frm a DNA template is known as: A)Replication B)Translation C)Transcripition D)Mutation Q3 Prokaryotic DNA –dependent RNA polymerase is a : A)Monomer B)Dimer C)Trimer D)Tetramer Q4. The enzyme required for transcription is: a) RNAase b) DNApolymerase c) RNApolymerase d) Restriction enzymes Q5 Mammalian RNA polymerase I synthesizes A) mRNA B) rRNA C) tRNA D) hnRNA Q6 Mammalian RNA polymerase III synthesizes A) mRNA B) rRNA C) tRNA D) hnRNA Q4. The enzyme required for transcription is: a) RNAase b) DNApolymerase c) RNApolymerase d) Restriction enzymes Q5 Mammalian RNA polymerase I synthesizes A) mRNA B) rRNA C) tRNA D) hnRNA Q6 Mammalian RNA polymerase III synthesizes A) mRNA B) rRNA C) tRNA D) hnRNA Q7:Which of the following transcription termination technique has RNA dependent ATPase activity? a)Intercalating agents b)Rho dependent c)Rho independent d)Rifampcin Q8 In mammals , synthesis of mRNA is catalysed by: A) RNA polymerase I B) RNA polymerase II C) RNA polymerase III D) RNA polymerase IV Q7:Which of the following transcription termination technique has RNA dependent ATPase activity? a)Intercalating agents b)Rho dependent c)Rho independent d)Rifampcin Q8 In mammals , synthesis of mRNA is catalysed by: A) RNA polymerase I B) RNA polymerase II C) RNA polymerase III D) RNA polymerase IV  References Essentials of Biochemistry, Pankaja Naik First Edition: 2012. Lippincott’s Illustrated Reviews: Biochemistry, 8th Edition. Textbook of biochemistry : with clinical correlations / edited by Thomas M. Devlin. - 7th ed.